Vol. 22, No. 8, 2009 / 1011
MPMI Vol. 22, No. 8, 2009, pp. 1011–1020. doi:10.1094/MPMI-22-8-1011.
Complete Genome Sequence
of Citrus Huanglongbing Bacterium,
‘Candidatus Liberibacter asiaticus’ Obtained
Yongping Duan,1 Lijuan Zhou,2 David G. Hall,1 Wenbin Li,3 Harshavardhan Doddapaneni,4 Hong Lin,4
Li Liu,5 Cheryl M. Vahling,1 Dean W. Gabriel,2 Kelly P. Williams,6 Allan Dickerman,6 Yijun Sun,5 and
1USDA-ARS-USHRL, Fort Pierce, FL 34945, U.S.A.; 2Dept. of Plant Pathology, University of Florida, Gainesville, FL 32653,
U.S.A.; 3USDA-APHIS-PPQ-CPHST, Beltsville, MD 20705, U.S.A.; 4USDA-ARS, Parlier, CA 93648, U.S.A.; 5ICBR, University
of Florida, Gainesville, FL, U.S.A.; 6Virginia Bioinformatics Institute Virginia Tech, Blacksburg, VA 24061, U.S.A.
Submitted 27 February 2009. Accepted 15 April 2009.
Citrus huanglongbing is the most destructive disease of cit-
rus worldwide. It is spread by citrus psyllids and is associ-
ated with a low-titer, phloem-limited infection by any of
three uncultured species of α α-Proteobacteria, ‘Candidatus
Liberibacter asiaticus’, ‘Ca. L. americanus’, and ‘Ca. L.
africanus’. A complete circular ‘Ca. L. asiaticus’ genome has
been obtained by metagenomics, using the DNA extracted
from a single ‘Ca. L. asiaticus’–infected psyllid. The 1.23-Mb
genome has an average 36.5% GC content. Annotation
revealed a high percentage of genes involved in both cell mo-
tility (4.5%) and active transport in general (8.0%), which
may contribute to its virulence. ‘Ca. L. asiaticus’ appears to
have a limited ability for aerobic respiration and is likely
auxotrophic for at least five amino acids. Consistent with its
intracellular nature, ‘Ca. L. asiaticus’ lacks type III and type
IV secretion systems as well as typical free-living or plant-
colonizing extracellular degradative enzymes. ‘Ca. L. asiati-
cus’ appears to have all type I secretion system genes needed
for both multidrug efflux and toxin effector secretion. Multi-
protein phylogenetic analysis confirmed ‘Ca. L. asiaticus’ as
an early-branching and highly divergent member of the
family Rhizobiaceae. This is the first genome sequence of an
uncultured α α-proteobacteria that is both an intracellular
plant pathogen and insect symbiont.
Citrus huanglongbing (HLB), popularly known as citrus
greening disease, was first noted in China in the early 20th
century (Zhao 1981). The disease is associated with three spe-
cies of phloem-restricted α-Proteobacteria, ‘Candidatus Liberi-
bacter asiaticus’ in Asia, ‘Ca. L. africanus’ in Africa (Jagoueix
et al. 1994), and ‘Ca. L. americanus’ in Brazil, South America
(Teixeira 2005). ‘Ca. L. asiaticus’ has a wide host range and
can infect, although not necessarily cause disease, on most
rutaceous species and some solanaceous species (Halbert and
Manjunath 2004). HLB is transmitted by two phloem-feeding
insect vectors, the Asian citrus psyllid Diaphorina citri and the
African citrus psyllid Trioza erytreae, and is considered the
most destructive disease of citrus in the world (Bove 2006;
Brlansky and Rogers 2007; Callaway 2008; Gottwald et al.
2007; Stokstad 2006). Management of the disease is not only
difficult but also expensive, and no cure is currently available
for infected trees. Approximately 100 million infected citrus
trees have been destroyed by the disease throughout Asia, with
an additional one million trees eliminated in Brazil since the
first report of the disease in São Paulo in 2004 (Gottwald et al.
2007). In the U.S., HLB was first discovered in August 2005 in
South Florida, seven years after the introduction of D. citri
into the state (Sutton et al. 2005). Since that time, HLB has
spread to all of Florida’s citrus-growing counties and has re-
cently been reported in the state of Louisiana (Southern Plant
Diagnostic Network 2008). Of even greater concern is the fact
that the vector D. citri can now be found in other citrus-pro-
ducing states, including Texas (Da Graça and Korsten 2004),
Hawaii, and California (Citrus Research Board 2008).
In addition to the Liberibacter sp. that is affecting the citrus
industry, a new Liberibacter species, ‘Ca. L. solanacearum’,
was recently associated with the emerging ‘zebra chip’ disease
of potatoes in the U.S. and tomatoes in New Zealand (Liefting
et al. 2008). ‘Ca. L. solanacearum’ is closely related to ‘Ca. L.
asiaticus’, although ‘Ca. L. solanacearum’ is not associated
with citrus HLB nor has it been found in Asian citrus psyllids
(Li et al. 2008b).
Attempts to obtain the entire Liberibacter genome using
HLB-affected plants have been unsuccessful, largely due to the
fact that the ‘Ca. L. asiaticus’ bacterium has not been cultured
and is present in very low titers in its hosts (the copy ratio of
‘Ca. L. asiaticus’ DNA to host plant genomic DNA is 1:1,000)
(Li et al. 2006). To date, only 24,477 nonredundant base pairs
David G. Hall, Wenbin Li, and Harshavardhan Doddapaneni contributed
equally to this work.
Current address for H. Doddapaneni: Department of Biology, Roy J.
Carver Center for Comparative Genomics, University of Iowa, Iowa City,
IA 52242, U.S.A.
Corresponding author: Y. Duan; E-mail: email@example.com
Nucleotide sequence data for the Whole Genome Shotgun project has been
deposited in the DDBJ/EMBL/GenBank database under project accession
number ABQW00000000. The version described in this paper is the first
*The e-Xtra logo stands for “electronic extra” and indicates that four sup-
plementary tables are published online.
This article is in the public domain and not copyrightable. It may be freely
reprinted with customary crediting of the source. The American Phyto-
pathological Society, 2009.
1012 / Molecular Plant-Microbe Interactions
(bp) from five contigs and two singlets (covering 12 full-length
genes, two partial genes, one pseudogene, and several inter-
genic regions of the bacterium) have been obtained from HLB-
affected plant DNA extracts by various methods (Hocquellet et
al. 1999; Jagoueix et al. 1994, 1997; Lin et al. 2008; Okuda et
al. 2005; Villechanoux et al. 1992). Another possible source of
‘Ca. L. asiaticus’ DNA is the insect vector. Despite the fact
that most infected psyllids collected from HLB-affected citrus
trees carry low titers of ‘Ca. L. asiaticus’, some heavily in-
fected adult psyllids harboring up to 1010 ‘Ca. L. asiaticus’
cells per head (Li et al. 2008a) can be found. Whole-genome
amplification methods, such as multiple displacement amplifi-
cation (MDA) (Dean et al. 2002), have been successfully ap-
plied to obtain whole insect genomes from a small amount of
initial DNA extract such as that found in first instar larval mos-
quitoes or single adult legs (Gorrochotegui-Escalante and
Black 2003). In this study, we report the first completed ge-
nome of the uncultured ‘Ca. L. asiaticus’ bacterium obtained
by metagenomics using MDA and pyrosequencing of the DNA
extracted from a single psyllid carrying a high titer of ‘Ca. L.
RESULTS AND DISCUSSION
Concentration of ‘Ca. L. asiaticus’ bacterial DNA
from a single psyllid.
Because of the low titer, phloem restriction, and uneven dis-
tribution of ‘Ca. L. asiaticus’ in all tested host plants, we chose
to obtain bacterial DNA from D. citri. In a D. citri population
from Florida, approximately 3% of the infected population
carried a ‘Ca. L. asiaticus’ population of approximately 108
bacterial cells per head, which was estimated to yield a 1:1
copy ratio of ‘Ca. L. asiaticus’ DNA to D. citri DNA (Li et al.
2008a). This ratio was approximately 1,000-fold higher than
that of ‘Ca. L. asiaticus’ DNA to plant DNA obtained from
symptomatic tissues of HLB-affected citrus plants. Based on
the linear regression of the absolute standard curve for ‘Ca. L.
asiaticus’ using real-time polymerase chain reaction (PCR) and
the fact that there are three copies of template 16S rDNA per
‘Ca. L. asiaticus’ genome, it was calculated that Psyllid #62
(Psy62) contained 1.29 × 1010 copies of the ‘Ca. L. asiaticus’
genome per microliter of DNA. Using multiplex PCR, the DNA
copy ratio of several possible sources of DNA were determined
(Fig. 1). The copy ratios of the main targets (D. citri, ‘Ca. L.
asiaticus’, and a Wolbachia sp.) were 1, 576, and 28, respec-
tively. Although the ‘Ca. L. asiaticus’ bacterium contains three
copies of 16S rDNA and the Wolbachia sp. contains only one
copy, the predominance of the ‘Ca. L. asiaticus’ DNA found in
the Psy62 sample would provide an adequate source for obtain-
ing the ‘Ca. L. asiaticus’ genome sequence.
Differentiation of ‘Ca. L. asiaticus’ genomic DNA
in the Psy62 sample.
After MDA amplification of Psy62 DNA and 454 pyrose-
quencing, a total of 419,571 shotgun reads (average read
length of 216 bp) were generated, containing 90,813,125 bp.
These shotgun reads were assembled into 1,475 contigs cover-
ing 2,386,844 bp. Each contig ranged in size from 500 to
186,240 bp. A total of 320 PCR confirmation reactions (cover-
ing approximately 551 kb of Psy62 DNA) were run against
both HLB-affected and unaffected samples obtained from cit-
rus, periwinkle, and psyllids (Fig. 2), to determine which of
the 1,475 contigs were associated with ‘Ca. L. asiaticus’ and
which were from other sources of DNA such as Wolbachia
spp. or D. citri. Ultimately, 38 contigs covering 1,225,539 bp
were confirmed by PCR to have originated from ‘Ca. L. asiati-
cus’. These ‘Ca. L. asiaticus’ contigs were assembled from
91,875 reads and covered 19,885,425 bp, providing an esti-
mated 16-fold coverage of the ‘Ca. L. asiaticus’ genome. Ex-
tensive PCR reactions that allowed the joining of HLB-associ-
ated contigs resulted in closure of the ‘Ca. L. asiaticus’ chro-
mosome. The gaps between these contigs were 853 bp on aver-
age, ranging from 24 to 2,885 bp. Of the 551 kb of confirma-
tion PCR DNA, 122,400 bp were resequenced and only 24 bp
were inconsistent with the original 454 assembly. Only three
misassemblies were discovered and Sanger sequencing was
performed to correct these misassembly errors. These indi-
cated that the Psy62 ‘Ca. L. asiaticus’ genome obtained by
MDA and 454 pyrosequencing had an accuracy ≥99.9% and
that the genomes among the population of ‘Ca. L. asiaticus’
cells in psyllid Psy62 were quite uniform.
of the Psy62 ‘Ca. L. asiaticus’ draft genome.
Recently, pulsed-field gel electrophoresis revealed the main
chromosome of the related species ‘Ca. L. americanus’ to be
circular and approximately 1.26 Mb in size (Wulff et al. 2008).
Consistent with these results, our ‘Ca. L. asiaticus’ genome
appears to be a single circular chromosome containing
1,227,204 bp, with an average GC content of 36.5% (Table 1).
Upon annotation, three operons for rRNA genes and a total of
44 tRNA genes were found within the genome. The replication
origin of the chromosome was predicted at position 436,457,
where the GC skew reached the global minimum. In the vicinity,
Fig. 1. Multiplex real-time polymerase chain reaction–based estimation of 16S rDNA copy numbers of the five major endosymbiotic bacteria from a single
Asian citrus psyllid (Diaphorina citri), namely, Arsenophonus sp., Carsonella ruddi, Wolbachia sp., Syn-symbiont, and ‘Candidatus Liberibacter asiaticus’.
Note the logarithmic scale.
Vol. 22, No. 8, 2009 / 1013
three DnaA boxes were arranged in inverted orientations. Ap-
proximately 74% of the 1,136 predicted coding sequences
have homologs with known and putative functions, while the
other 26% represent hypothetical conserved open reading
frames, which are conserved in other microorganisms but with
unknown function. A total of 32 genes were determined to be
pseudogenes. Table 2 contains a summary of the COG (Clus-
ters of Orthologous Groups database) assignments and their
functional categories. As with most reduced genomes, there
were a relatively low number of genes involved in the biosyn-
thesis of compounds readily taken up from the host and regula-
tory elements, including σ factors, probably reflecting the
reduced environmental challenges associated with an intracel-
lular lifestyle (Konstantinidis and Tiedje 2004). By contrast,
there were a high number of genes, particularly for a small
genome, involved in cell motility (4.5% in ‘Ca. L. asiaticus’ as
compared with 0.1% in the Wolbachia sp. and 1.2% in α-Pro-
teobacteria [Table 2]), including type IV pili and flagellar
genes. There were 92 genes involved in active transport, in-
cluding 40 ATP-binding cassette (ABC) transport genes. In
contrast to the Wolbachia sp., no transposons or insertion ele-
ments were identified in the ‘Ca. L. asiaticus’ genome (Wu et
al. 2004). However, 12 phage-related genes were found, indi-
cating that a phage or prophage may be present.
Phylogenetic analysis and comparative genomics.
The ‘Ca. L. asiaticus’ genome from Pys62 was incorporated
into a robust species tree for α-Proteobacteria, based on 104
conserved genes that show no evidence of horizontal transfer
(Williams et al. 2007). Two of these 104 genes were not identi-
fied in ‘Ca. L. asiaticus’, and to ensure that only vertically
transmitted genes were used, an additional eight genes were
rejected that scored poorly in a hidden Markov model (HMM)
for the family. Bayesian phylogenetic analysis of the concate-
nation of the trimmed alignments for the remaining 94 proteins
produced a tree with identical topology and similar lengths to
that obtained previously, with 100% bootstrap support for
every branch (Fig. 3). The current analyses using robust ge-
nome-wide, gene-based phylogeny places ‘Ca. L. asiaticus’
within the order Rhizobiales in agreement with the previous
16S and other gene-based analyses (Doddapaneni et al. 2008;
Williams et al. 2007). Furthermore, for the first time and using
large samples of both bacteria and genes, we are able to spe-
cifically show that within the order Rhizobiales, ‘Ca. L. asiati-
cus’ is closely associated with members of the Rhizobiaceae
family. Based on its position in the tree, which received 100%
Bayesian support, ‘Ca. L. asiaticus’ can be considered an
early-branching member of the Rhizobiaceae family. It is inter-
esting to note that the distance from the root is far longer for
‘Ca. L. asiaticus’ than for any other tested member of the order
Rhizobiales (indeed, exceeded among all α-Proteobacteria
only by Neorickettsia sennetsu), suggesting that it has evolved
much more rapidly. Such rapid genome evolution is typical of
host-restricted symbionts or pathogens, usually explained by
elevated genetic drift resulting both from population bottlenecks
and relaxed selection on many genes (Moran et al. 2008).
Analysis of the putative protein coding gene sequences of
Psy62 ‘Ca. L. asiaticus’ (1,136 proteins) with the genomes of
other related bacteria supports phylogenetic positioning of this
bacterium rather than positioning by a niche-based genome
Fig. 2. Polymerase chain reaction (PCR) confirmation of the ‘Candidatus Liberibacter asiaticus’ genome sequences using infected vs. noninfected citrus,
periwinkle, and psyllids. Lane M, 1-kb DNA ladder; lane 1, Citrus huanglongbing (HLB)-affected citrus from field; lane 2,HLB-affected citrus from green
house; lane 3, HLB-free citrus from field; lane 4, HLB-free citrus from green house; lanes 5 and 6, HLB-affected periwinkles from greenhouse; lane 7, HLB-
free periwinkle from green house; lane 8, pooled DNA from eight individual ‘Ca. Liberibacter asiaticus’–infected psyllids; lane 9, pooled DNA from 10
individual ‘Ca. Liberibacter asiaticus’–free psyllids; and lane 10, no DNA negative control. Numbers above PCR reactions beginning with LJ correspond to
the primer pairs used for each PCR reaction.
Table 1. General features of ‘Candidatus Liberibacter asiaticus’ genome
Feature Corresponding no.
Total number of predicted genes
Protein coding genes
Genes assigned to the COG (Clusters of
Orthologous Groups) database
1014 / Molecular Plant-Microbe Interactions
adaptation. A four-way comparison of the ‘Ca. L. asiaticus’
genome was performed with the genomes of Sinorhizobium
meliloti (3,341 proteins), Candidatus Phytoplasma asteris (754
proteins), and Wolbachia pipientis (1,208 proteins) (Fig. 4). A
total of 174 proteins were present in all four genomes, with a
majority of these proteins playing a role in the core metabolic
pathways of the cell. There were 7 to 255 proteins present in
three of the four genomes compared, although no common
proteins were found among the other three genomes if S.
meliloti was excluded. In this analysis, one protein (Na+/H+-
dicarboxylate symporter) was found to be common only be-
tween W. pipientis and ‘Ca. L. asiaticus’ (both are insect sym-
bionts) but none of the other genomes, while no common pro-
teins existed only between ‘Ca. P. asteris’ and ‘Ca. L. asiaticus’
(the two phloem pathogens and insect endosymbionts). On the
other hand, 279 proteins (24.8%) present in ‘Ca. L. asiaticus’
are also present only in S. meliloti (two closely related mem-
bers of the Rhizobiaceae family).
In order to provide a framework from which the metabolic
needs of the organism could be deduced, all major metabolic
pathways and enzyme reactions predicted from the ‘Ca. L. asi-
aticus’ genome sequence were mapped onto established meta-
bolic pathways and are summarized here. Briefly, ‘Ca. L. asi-
aticus’ contains all 14 genes that typically encode NADH
dehydrogenase subunits [A-N], a major component of the res-
piratory electron transport chain. However, no homologs corre-
sponding to the terminal stages of oxidative phosphorylation
were identified. This includes the absence of two key enzymes,
polyphosphate kinase (EC 184.108.40.206) and inorganic diphospha-
tase (EC 220.127.116.11), that metabolize pyrophosphate to Pi and
ADP. Likewise, the ‘Ca. L. asiaticus’ genome does not appear
to have homologs for the cytochrome bc1 complex (EC
18.104.22.168), cytochrome c oxidase, the cbb3-type (EC 22.214.171.124),
or the cytochrome bd complex although all four cytochrome O
ubiquinol oxidase subunits [I to IV] have been identified. This
is interesting because α-Proteobacteria (the class containing
‘Ca. L. asiaticus’) typically employ cytochrome c oxidases as
terminal oxidases, whereas γ-Proteobacteria (the class contain-
ing E. coli and Xylella fastidiosa) employ quinol oxidases
(Foster et al. 2005). Unlike E. coli, which has two terminal
oxidases (Anraku and Gennis 1987), the respiratory complex
of ‘Ca. L. asiaticus’ resembles that of the citrus pathogen X.
fastidiosa (Bhattacharyya et al. 2002), which has one terminal
oxidase encoded for by the cyo operon and is active only under
oxygen-rich conditions (Cotter et al. 1990; Rice and Hempfling
1978). Therefore, due to the lack of key enzymes involved in
oxidative phosphorylation as well as absence of diverse termi-
nal oxidases, it can be concluded that ‘Ca. L. asiaticus’ has a
limited capacity for aerobic respiration.
Because of the limited aerobic capabilities, pathways neces-
sary for anaerobic respiration were also investigated. The ‘Ca.
L. asiaticus’ genome did not contain any genes involved in sul-
fur metabolism; however, several enzymes involved in nitro-
gen metabolism, such as a NAD+ synthase (EC 126.96.36.199), gluta-
Table 2. Summary of COG (Clusters of Orthologous Groups) database assignments by functional category and correlation with the total number of open
Functional class and categories No. of genes % of total Correlationa % in Wolbachia AE017321 % in α α-proteobacteriab
Information storage and processing
J Translation, ribosomal structure &
L DNA replication, recombination,
D Cell division and chromosome
V Defense mechanisms
O Postranslational modification
M Cell envelope biogenesis, outer
P Inorganic ion transport and
U Intracellular trafficking and secretion
N Cell motility
T Signal transduction
F Nucleotide transport and metabolism
G Carbohydrate transport and
E Amino acid transport and metabolism
H Coenzyme metabolism
I Lipid metabolism
C Energy production and conversion
Q Secondary metabolite transport and
R General function prediction only
S Function unknown
Not in COG
25 2.20 No 4.02 4.79
a The expected positive (+), negative (–), or no correlation (No) of genome size with the number of genes in each COG assignment category (Konstantinidis
and Tiedje 2004).
b Representative small (940 genes encoded by 1.08 Mb) α-Proteobacteria genome.
Vol. 22, No. 8, 2009 / 1015
mine synthetase (EC 188.8.131.52), and glutaminase (EC 184.108.40.206),
along with those enzymes involved in the glutamate metabo-
lism were identified, suggesting a dependence of ‘Ca. L. asi-
aticus’ on nitrogen metabolism. The above results imply that
‘Ca. L. asiaticus’ can not reduce sulfate and possibly depends
heavily upon nitrogen utilization to generate energy through
Key enzymes such as PfkA, which encodes for 6-phospho-
fructokinase (EC 220.127.116.11) in bacteria (Koonin and Galperin
2003), and Pgm, which encodes a phosphoglucomutase (EC
18.104.22.168), were present in the genome, while no enzymes for the
Entner-Doudroff pathway were uncovered, indicating that
glycolysis is the major pathway for the catabolism of mono-
saccharides. Based on the enzymes that are present, ‘Ca. L.
asiaticus’ has the ability to metabolize sugars such as glucose,
fructose, and xylulose but not mannose, galactose, rhamnose,
or cellulose. How these sugars are transported into the cell
remains unclear, since the only phosphotransferase system
(PTS) protein identified within the genome was a SgaT ho-
molog, a component of a PTS permease. Considering the mul-
tiple ABC transporter proteins in the ‘Ca. L. asiaticus’ genome,
it is highly possible that these may be involved in sugar trans-
port. Several obligate intercellular parasites, such as Chlamy-
dia trachomatis and Rickettsia prowazeki, have developed a
unique system for obtaining energy. These bacteria can act as
energy parasites and scavenge ATP from their host through the
use of an ATP/ADP translocase (Hatch et al. 1982; Winkler
1976). Interestingly, ‘Ca. L. asiaticus’ encodes for an
ATP/ADP translocase in addition to its ATP synthase, allowing
it to both synthesize ATP as well as uptake this energy source
directly from its surroundings. Additionally, genes for the tri-
carboxylic acid (TCA) cycle, including a gene encoding for
citrate synthase (gltA), the principal port of entry of acetyl-
Fig. 3. Consensus tree from Bayesian phylogenetic analysis based on 94 proteins. Branches for the 46 strains most distant to ‘Candidatus L. asiaticus’ have
been removed for clarity. All nodes had 100% support.
Fig. 4. Venn diagram comparing homologous and unique proteins among
‘Candidatus L. asiaticus’, Wolbachia pipientis (an insect symbiont),
Sinorhizobium meliloti (a member of the Rhizobiaceae family), and ‘Ca. P.
asteris’ (a phloem pathogen and insect symbiont).
1016 / Molecular Plant-Microbe Interactions
CoA into the TCA cycle that is often used as an indicator of
the presence of a functional TCA cycle, were identified in ‘Ca.
L. asiaticus’. Given the presence of a functional TCA cycle,
‘Ca. L. asiaticus’ may utilize a range of amino acids as energy
sources. These include glutamate, alanine, aspartate, glycine,
serine, threonine, methionine, cysteine, arginine, proline, his-
tidine, tyrosine, phenylalanine, and tryptophan. With the ex-
ception of proline and tryptophan, all other amino acids listed
above have been identified as components of the phloem sap in
other studies (Ohshima et al. 1990), and thus, sap may act as
the primary source of these amino acids for ‘Ca. L. asiaticus’.
Enzymes involved in the conversion steps from metabolic
intermediates to the synthesis of amino acids have been identi-
fied in ‘Ca. L. asiaticus’ for only two candidates, phenylpyru-
vate to phenylalanine and asparate to lysine. Those involved in
the synthesis of tryptophan, tyrosine, leucine, isoleucine, and
valine from metabolic intermediates are absent. Three of the
above amino acids are known to be present in phloem, which
is probably the direct source of these amino acids, while the
source of the other two amino acids, tryptophan and valine,
has yet to be determined.
One of the enzymes in the pentose phosphate pathway
(PPP), known as transaldolase (EC 22.214.171.124), is absent in ‘Ca.
L. asiaticus’. However, ‘Ca. L. asiaticus’ has other enzymes of
the PPP and the full pathways for both purine and pyramidine
biosynthesis, suggesting the existence of a functional but diver-
gent form of this enzyme in ‘Ca. L. asiaticus’. Previous studies
have shown that transaldoase exists as several uncharacterized
isoforms in bacterial genomes (Kube et al. 2008), thus coin-
ciding with our hypothesis.
Transport proteins and types of secretion systems.
Similarity searches of the 1,136 ‘Ca. L. asiaticus’ proteins
classified 137 as transporter proteins. These 137 proteins fall
into five of the nine known classification categories. Of these
proteins, nine proteins belong to the channels/pores class of
transporters, 24 proteins belong to the electrochemical poten-
tial-driven transporters, 92 are classified under primary active
transporters, one belongs to the group translocators class, and
the remaining 11 proteins can be classified under the incom-
pletely characterized transport systems class of transporters.
Of the 92 primary active transporters, 40 are ABC transporters,
which is typical of α-Proteobacteria with a wide host range but
in stark contrast to intracellular bacteria of a similar size, in
which the average is 15 ABC transporters (Davidson et al.
2008). It is quite possible that some of these transporters affect
virulence, host range, or symptom elicitation, alone or in com-
bination. For example, the phosphate transport system found in
‘Ca. L. asiaticus’ utilizes an ABC-type transporter that is
known to mediate the uptake of phosphate into the cell and has
been reported in several studies to be associated with the viru-
lence of the bacteria (Daigle et al. 1995; Mantis and Winans
1993; von Kruger et al. 1999). Another example of an ABC
transporter required for virulence in other organisms is the
zinc transport system (znuABC) (Garrido et al. 2003). The
presence of znuABC genes in ‘Ca. L. asiaticus’ may allow the
bacteria to uptake this micronutrient from the phloem, result-
ing in a local zinc deficiency and, thus, the mimicry of the
symptoms between HLB-affected and zinc-deficient plants.
Not surprisingly for an intracellular bacterial pathogen
whose route of infection involves direct injection by the psyl-
lid into host cells, no type III or IV secretion systems or their
effectors, including avirulence genes, were found. Type III and
IV systems can play key offensive roles in allowing extracel-
lular pathogens to attack both plant and animal hosts (Fauvart
and Michiels 2008; Felix et al. 2008; Munkvold et al. 2008).
Also not found in the genome were plant cell-wall degrada-
tion enzymes such as cellulases, pectinases, xylanases, or en-
doglucanases, which require type II secretion. In addition,
genes typically involved in the external secretion of extracel-
lular enzymes as part of the main terminal branch (MTB) of
the type II secretion system (Johnson et al. 2006) were not
However, all proteins required for the first step of the type II
secretion system, the general secretory pathway (TC 3.A.5.),
which is responsible for the export of proteins to the periplasm
(Pugsley 1993), were found in the ‘Ca. L. asiaticus’ genome
from Psy62. In addition, 10 putative proteins required for pilin
secretion and assembly as part of the MTB (TC 3.A.15) were
found, indicating the potential for type IV pilus secretion and
assembly. Four putative pilin subunit (COG3847) proteins
were also found. Although pilus formation by ‘Ca. L. asiati-
cus’ needs to be experimentally verified, if present, the type IV
pili could be used for autoaggregation and biofilm formation,
as in Xylella spp., in which it is responsible for the obstruction
of xylem flow (De La Fuente et al. 2008).
A nearly complete set of 30 genes involved in flagella bio-
synthesis were also found in the ‘Ca. L. asiaticus’ genome
(Supplementary Table S1). However, despite extensive pub-
lished electron microscopy of ‘Ca. L. asiaticus’ in both citrus
and psyllid hosts (Bove 2006), there is no evidence to our
knowledge that ‘Ca. L. asiaticus’ cells are flagellated. One
gene encoding the flagella motor switch protein (fliN) and
three genes encoding the flagella motor protein and chemo-
taxis motility protein (motB, motC, and motD) appear to be
pseudogenes (Supplementary Table S2). This, perhaps, may be
the reason for the lack of an observable structure.
Several complete type I secretion systems were present. Two
primary functions for the type I secretion machinery have been
elucidated. The first one is defensive, involving multidrug
efflux, protecting the bacterium against toxic environmental
chemicals, antibiotics produced by other bacteria, and phyto-
alexins produced by hosts. Multidrug efflux has been demon-
strated as an important mechanism for bacterial survival in
members of genera Erwinia, Rhizobium, Agrobacterium, Bra-
dyrhizobium, and Xanthomonas (Reddy et al. 2007). The other
function is offensive, allowing the secretion of a variety of
degradative enzymes and offensive effectors, some of which
are antibiotics and others involved in plant or animal patho-
genicity. Offensive enzymes and effectors known to be secreted
via the type I system include a limited number of hydrolases
(proteases, phosphatases, esterases, nucleases, and glucanases)
and a relatively large number of protein toxins, including RTX
hemolysins and bacteriocins (Delepelaire 2004; Koronakis et
al. 2004). In gram-negative bacteria, type I secretion systems
are typically composed of three protein components, two of
which are localized in the inner membrane and one, TolC, that
traverses both the periplasm and outer membrane (Koronakis
et al. 2004). Although most phytopathogenic bacteria possess
multiple copies of tolC, only one copy was found in the ‘Ca.
L. asiaticus’ genome. Interestingly, only one copy of tolC is
also found in each of the Xylella genomes (Reddy et al. 2007).
Two type I defensive system gene fusions were found in ‘Ca.
L. asiaticus’, both in COG1132: i) ABC-type multidrug ex-
porter family fused ATPase and ii) inner membrane subunits.
In addition, one complete type I offensive system was found,
with both genes in close proximity, i.e., COG4618 (ATPase)
and COG0845 (membrane fusion protein). A putative type I
effector, a hemolysin (COG1253), was found close to the two
type I offensive system genes. Since knockouts of the single
tolC gene in Xylella spp. are completely nonpathogenic and
highly sensitive to phytoalexins (Reddy et al. 2007), this raises
Vol. 22, No. 8, 2009 / 1017
the possibility of a chemical- or gene-engineered approach to
attack the single ‘Ca. L. asiaticus’ target.
Successful plant pathogens have long been thought to have
the ability to avoid eliciting plant defense responses, to ac-
tively suppress plant defenses, or both, mainly through effec-
tors secreted by a type III system (Abramovitch and Martin
2004). Given the relatively reduced genome size of ‘Ca. L. asi-
aticus’ and the lack of a type III secretion system, ‘Ca. L. asi-
aticus’ may primarily rely upon an avoidance strategy. The
lack of type II plant cell-wall degradative enzymes avoids the
problem of eliciting defense responses based on autodegrada-
tion products of the plant cell wall (oligogalacturonides)
(Orozco-Cardenas and Ryan 1999). On the other hand, it is
more difficult to see how ‘Ca. L. asiaticus’ can avoid eliciting
plant recognition of nonself, pathogen-associated molecular
patterns (PAMPs), particularly of lipopolysaccharide (LPS)
and LPS fragments (Braun et al. 2005) and possibly also of
flagellin and flagellin fragments (Gomez-Gomez et al. 1999;
Marutani et al. 2005). ‘Ca. L. asiaticus’ encodes 57 genes in
COG functional category M (cell envelope biogenesis and
outer membrane, including LPS), which is 5% of the total and
approximately what would be expected for a genome of this
size (Table 2). Therefore LPS fragments may elicit a PAMP
response, even if flagellin or flagellin fragments are not pro-
duced. This raises the intriguing possibility that ‘Ca. L. asiati-
cus’ secrets a non–type III suppressor of host defense. Indeed,
spiroplasmas and phytoplasmas, which do not have type III
systems, are also injected into the cytoplasm of phloem cells
by their insect vectors and secrete proteins into the phloem cell
cytoplasm, allowing movement of these proteins to other plant
cells via plasmodesmata (Hogenhout and Loria 2008). One
such protein is a plant gene-regulating effector, SAP11, which
contains a nuclear localization signal that is functional in plant
cells (Hogenhout et al. 2008).
HLB symptoms and ‘Ca. L. asiaticus’ bacterial titer vary
among infected plants. Some disease symptoms, such as yellow
shoots, chlorosis, and vein corking are nondescript and difficult
to distinguish from nutrient deficiencies. There is no indication
that ‘Ca. L. asiaticus’ actively conditions pathogenicity, since
analysis of the genome revealed no toxins, enzymes, or special-
ized secretion systems. Instead, the intracellular lifestyle of ‘Ca.
L. asiaticus’ is better described as parasitic rather than patho-
genic, with disease symptoms arising primarily as a result of
host metabolic imbalances caused by ‘Ca. L. asiaticus’ nutrient
depletion or interference of transportation. Infection by ‘Ca. L.
asiaticus’ leads to the plugging of sieve pores, primarily by cal-
lose deposition. The phloem blockage or damage then leads to
massive accumulation of starch in leaves and nutrient deficien-
cies in sink organs (Kim et al. 2009). How ‘Ca. L. asiaticus’ is
able to infect such a wide variety of citrus plants and their rela-
tives is currently under investigation.
Because of the rapid spread and threat of HLB to the citrus
industries in the world, efforts are being made to decipher the
genetic information of the HLB bacterium ‘Ca. L. asiaticus’.
Due to its fastidious nature, however, this bacterium remains
unculturable, representing a major obstacle towards the ad-
vancement of the field over the past century. In spite of this
limitation, we have obtained and annotated the entire genome
of ‘Ca. L. asiaticus’, using MDA and 454 pyrosequencing
technologies on DNA extracted from a single ‘Ca. L. asiati-
cus’–infected Asian citrus psyllid (D. citri). This is the first
genome sequence of an uncultured α-Proteobacteria that acts
as both an intracellular plant pathogen and an insect symbiont.
It is important to note that ‘Ca. L. asiaticus’ contains genetic
features distinctive to obligate intracellular bacteria (Moran
2002), such as having a small genome size (1.23 Mb for ‘Ca.
L. asiaticus’), a low GC content (36.5% for ‘Ca. L. asiaticus’),
and a significant genome reduction compared to other mem-
bers of the Rhizobiaceae family. The information revealed by
the completed genome may make it possible to identify those
conditions necessary for its growth as well as aid in our under-
standing of how this pathogen becomes established in both its
vector and plant hosts. This genome may also provide infor-
mation relevant to two other genomes of HLB bacteria, ‘Ca. L.
americanus’ and ‘Ca. L. africanus’, thus lending insight into
measures to control this devastating disease and sustain the cit-
MATERIALS AND METHODS
All experiments using live D. citri were performed in the
insect-proofed greenhouse in the United States Horticulture
Research Laboratory, United States Department of Agriculture
(USDA)-Agricultural Research Service (Fort Pierce, FL,
U.S.A.). Clean adult psyllids maintained in cages were trans-
ferred to a cage with an HLB-affected lemon plant (Citrus
limon) with a high titer of ‘Ca. L. asiaticus’ bacterium. After
feeding for 45 days on the infected plant, 15 psyllids were col-
lected, using an aspirator, and were stored in 95% ethanol for
DNA extraction from psyllids.
DNA from individual psyllids was extracted as described
(Hung et al. 2004). Briefly, individual psyllids were air-dried
for 10 min., were homogenized in a 1.5-ml tube containing
300 μl of extraction buffer (100 mM Tris-HCl, pH 8.0, 50 mM
EDTA, 500 mM NaCl, 1% N-lauroylsarcosinel), and were
incubated at 55
form/isoamyl alcohol (25:24:1) was then added to each tube,
and the tube was vortexed and centrifuged at 12, 000 × g, 4°C
for 10 min. The supernatant (approximately 200 μl) was trans-
ferred to a new 1.5-ml tube containing 500 μl of 100% ethanol,
which was gently inverted several times and was centrifuged at
14,000 × g, 4°C for 10 min. The resulting DNA pellet was
washed once with 70% ethanol. The pellet was resuspended in
15 μl of water and was stored at –20°C. All extractions were
performed in a laminar flow hood to avoid contamination.
oC for 1 h. An equal volume of phenol/chloro
Quantitative TaqMan real-time PCR amplifications were
performed with the specific primers and probes (Supplementary
Table S3), using a SmartCycler II (Cepheid, Sunnyvale, CA,
U.S.A.) in a 25-μl reaction volume, according to the standard-
ized conditions and program (Li et al. 2006). Reactions used
1μl of the original DNA extracts obtained directly from in-
fected psyllids or 2 μl of the MDA-generated DNA obtained as
described above. To estimate DNA template concentrations,
absolute standard curves were established using the plasmid
DNA cloned with PCR amplicons from each template, as de-
scribed previously (Li et al. 2008c). The data were analyzed
using SmartCycler software version 2.0D.
DNA extracted from Psy62 was used as a template for
MDA, using the REPLI-g whole genome amplification kit
(Qiagen, Germantown, MD, U.S.A.). Briefly, 40 ng of DNA
was subjected to whole genome amplification in a 50-μl reac-
tion by random priming and strand-displacement synthesis
30°C, according to the manufacturer’s recommendations. After
an overnight incubation, the reaction was terminated by incu-
1018 / Molecular Plant-Microbe Interactions
obtained from a MDA reaction as estimated by a NanoDrop
1000 spectrophotometer (Wilmington, DE, U.S.A.). DNA was
stored at –20°C.
at 65°C for 3 min. Approximately 8.5 μg of DNA was
Sequencing and assembly.
Initially, 454 pyrosequencing and sequence assemblies were
conducted by the Interdisciplinary Center for Biotechnology
Research, University of Florida, Gainesville. DNA sequencing
was performed as described by Margulies and associates
(2005), with slight modifications as specified by 454 Life Sci-
ences (Roche, Branchburg, NJ, U.S.A.). Briefly, high molecu-
lar–weight DNA was sheared by nebulization to a size range of
300 to 800 bp. DNA fragment ends were repaired and phos-
phorylated using T4 DNA polymerase and T4 polynucleotide
kinase. Adaptor oligonucleotides ‘A’ and ‘B’ supplied with the
454 Life Sciences sequencing reagent kit were ligated to the
DNA fragments using T4 DNA ligase. Purified DNA fragments
were hybridized to DNA capture beads and were clonally am-
plified by emulsion PCR. DNA capture beads containing am-
plified DNA were deposited in individual regions of a 70 × 75
mm PicoTiter plate, and DNA sequences were determined
using the GS-FLX instrument. DNA sequence information
from the initial and supplementary runs was combined in a
single assembly using Newbler sequence assembly software
Sequence validation and annotation.
To confirm that the assembled contigs were correct, 320 sets
of confirmation primers (Supplementary Table S4) were de-
signed based on the contig sequences predicted to be from
‘Ca. L. asiaticus’, each set yielding an average PCR product of
1,900 bp. DNA from HLB-affected and HLB-free citrus, peri-
winkle, and psyllid samples was used to validate the target
DNAs and assembled contigs. PCR products of the expected
size (at least one amplified product from each contig) were
combined from all positive samples and were purified and
resequenced to confirm their sequence identity. After confir-
mation, the ‘Ca. L. asiaticus’ sequences were annotated using
the National Center for Biotechnology Information (NCBI)
annotation pipeline with GeneMarkS (Besemer et al. 2001)
program and the FGENESB software (SoftBerry Inc., Mount
Kisco, NY, U.S.A.). Briefly, protein coding (mRNA) genes
were identified using Markov chain models with self-trained
parameters. Their functions were assigned by similarity
searches against the NCBI COG database (Tatusov et al. 1997)
and Conserved Domain Database (Marchler-Bauer et al.
2002). Potential rRNA genes were identified by BLAST
against bacterial rRNA databases. tRNAs were predicted using
tRNAscan-SE (Lowe and Eddy 1997). Ribosome binding sites,
promoters, and terminators prediction information was used as
evidences to identify potential operons. Automatic annotations
were stored in the myCAP system, a web application with
database backend, for review and manual curation (available
Comparative genomics and contig gap closure.
A BLASTP search of predicted ‘Ca. L. asiaticus’ proteins
was followed by mapping the contigs representing predicted
orthologs using the genomes of two phylogenetically related
organisms, Sinorhizobium meliloti and Bartonella quintana, as
potential scaffolds using Circos software (available online).
After mapping the contigs, end primers were designed and
used to amplify Psy62 DNA. Amplified DNA was then se-
quenced and was used to close gaps of the chromosome.
Bayesian phylogenetic analysis. Orthologs of ‘Ca. L. asiati-
cus’ were identified for 102 of the 104 conserved proteins (all
except CcmC and MnmA) that were used in a previous phy-
logenetic tree constructed for 72 α-Proteobacteria and eight
non–α-Proteobacteria strains (Williams et al. 2007). Each ‘Ca.
L. asiaticus’ ortholog was ranked relative to those for the other
80 species using the HMM score, with a median ranking of
12th worst, and eight protein families (UvrD, UbiA, UvrA,
YaeL, LigA, RpsH, RpsO, and AlaS) for which the ‘Ca. L. asi-
aticus’ proteins scored no better than third worst were rejected.
Sequences for the remaining 94 protein families were aligned,
and ambiguous portions of the alignments were removed to
prepare a concatenated protein sequence with 23,219 amino
acids. Three single-chain 800,000-generation MrBayes (avail-
able online) runs were performed, and all trees from the latter
400,000 generations showed identical topology and were used
to prepare a consensus tree.
In silico analysis of proteins involved
in metabolism and transport.
Reconstruction of predicted ‘Ca. L. asiaticus’ metabolic
pathways was carried out using IdentiCS software (Sun and
Zeng 2004). Annotated enzyme models were used to map the
‘Ca. L. asiaticus’ data onto the KEGG (Kyoto Encyclopedia of
Genes and Genomes) metabolic pathways (Kanehisa and Goto
2000; Kanehisa et al. 2006, 2008). A comparison between ‘Ca.
L. asiaticus’ proteins and proteins in the Transporter Classifi-
cation database of the Saier Lab Bioinformatics Group (avail-
able online) was made to classify and group transporter pro-
teins in the newly annotated genome according to the widely
accepted Transporter Classification system.
The authors thank P. Li and J. Mozoruk for technical support and L.
Benyon for critically reviewing this paper. Funding for this work was pro-
vided by the Florida Citrus Production Research Advisory Council, Flor-
ida Department of Agriculture and Consumer Services, contract 013644.
Funding for D. W. Gabriel was provided by USDA-APHIS.
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AUTHOR-RECOMMENDED INTERNET RESOURCES
University of Florida Biotechnology Vision server myCAP system:
Genome Sciences Centre’s Circos software: mkweb.bcgsc.ca/circos
MrBayes software: mrbayes.csit.fsu.edu
Saier Lab Bioinformatics Group’s Transporter Classification database: