Living with Genome Instability: the Adaptation of Phytoplasmas toDiverse Environments of Their Insect and Plant Hosts††
Xiaodong Bai,1 Jianhua Zhang,1,2† Adam Ewing,1‡ Sally A. Miller,2 Agnes Jancso Radek,3§Dmitriy V. Shevchenko,3 Kiryl Tsukerman,3 Theresa Walunas,3 Alla Lapidus,3¶
John W. Campbell,3� and Saskia A. Hogenhout1*Department of Entomology1 and Department of Plant Pathology,2 The Ohio State University, Ohio Agricultural Research and
Development Center, Wooster, Ohio 44691, and Integrated Genomics, Chicago, Illinois 606123
Received 30 September 2005/Accepted 23 January 2006
Phytoplasmas (“Candidatus Phytoplasma,” class Mollicutes) cause disease in hundreds of economically importantplants and are obligately transmitted by sap-feeding insects of the order Hemiptera, mainly leafhoppers andpsyllids. The 706,569-bp chromosome and four plasmids of aster yellows phytoplasma strain witches’ broom(AY-WB) were sequenced and compared to the onion yellows phytoplasma strain M (OY-M) genome. The phyto-plasmas have small repeat-rich genomes. This comparative analysis revealed that the repeated DNAs are organizedinto large clusters of potential mobile units (PMUs), which contain tra5 insertion sequences (ISs) and genes forspecialized sigma factors and membrane proteins. So far, these PMUs appear to be unique to phytoplasmas.Compared to mycoplasmas, phytoplasmas lack several recombination and DNA modification functions, and there-fore, phytoplasmas may use different mechanisms of recombination, likely involving PMUs, for the creation ofvariability, allowing phytoplasmas to adjust to the diverse environments of plants and insects. The irregular GCskews and the presence of ISs and large repeated sequences in the AY-WB and OY-M genomes are indicative of highgenomic plasticity. Nevertheless, segments of �250 kb located between the lplA and glnQ genes are syntenic betweenthe two phytoplasmas and contain the majority of the metabolic genes and no ISs. AY-WB appears to be furtheralong in the reductive evolution process than OY-M. The AY-WB genome is �154 kb smaller than the OY-Mgenome, primarily as a result of fewer multicopy sequences, including PMUs. Furthermore, AY-WB lacks genes thatare truncated and are part of incomplete pathways in OY-M.
Phytoplasmas cause disease in over 200 economically impor-tant plants and are obligately transmitted by phloem-feedinginsects of the order Hemiptera, mainly leafhoppers and psyl-lids. They are unique bacteria, as they can efficiently invade
cells of insects and plants, organisms belonging to two king-doms. Phytoplasmas are members of the class Mollicutes. Mol-licutes are soft-skinned (mollis, soft; cutis, skin [in Latin]) bac-teria due to the lack of an outer cell wall and usually have smallgenomes, a low G�C content, few rRNA operons, few tRNAgenes, and limited metabolic activities (18). Mollicutes repre-sent a branch of the phylogenetic tree of the gram-positiveeubacteria and are most closely related to low-GC gram-pos-itive bacteria such as Bacillus, Clostridium, and Streptococcusspp. (94, 96).
The phylogenetic tree of mollicutes is composed of twomajor clades that diverged early in evolution (51). One cladecontains the orders Acholeplasmatales and Anaeroplasmatales(AAA clade mollicutes), and the other clade contains the or-ders Mycoplasmatales and Entomoplasmatales (SEM clade molli-cutes) (9). Phytoplasmas, formerly known as mycoplasma-likeorganisms of plants, form a monophyletic group in the orderAcholeplasmatales (51) and were recently assigned to a novelgenus, “Candidatus Phytoplasma” (41). Approximately 20 phy-toplasma phylogenetic groups have been proposed based on16S rRNA gene sequences, and new branches are continuouslybeing discovered (69, 85). Members of the order Acholeplas-matales are distinct from other mollicutes in several ways. Forinstance, whereas most mollicutes use UGA as a tryptophancodon instead of a stop codon, a feature they share with mi-tochondria, the acholeplasmas and phytoplasmas retainedUGA as a stop codon (80).
Mollicutes have been extensively studied because of theireconomic importance. They are disease agents and obligate
* Corresponding author. Mailing address: Department of Entomology,The Ohio State University, Ohio Agricultural Research and DevelopmentCenter, 1680 Madison Avenue, Wooster, OH 44691. Phone: (330) 263-3730. Fax: (330) 263-3686. E-mail: [email protected].
† Present address: Potato Research Center, Agriculture and Agri-Food Canada, Fredericton, NB E3B 4Z7, Canada.
‡ Present address: GCB Graduate Group, University of Pennsylva-nia, Philadelphia, PA 19104.
§ Present address: Epicentre Technologies Corp., Madison, WI 53713.¶ Present address: Microbial Genomics, DOE Joint Genome Insti-
tute, Walnut Creek, CA 94598.� Present address: Scarab Genomics, LLC, Madison, WI 53713.†† Authors’ contributions: X.B., performance of the majority of the
bioinformatics analysis (annotation, comparative genome analyses, de-fining metabolic pathways of AY-WB, and submission of sequences toGenBank), and writing of manuscript; J.Z., development of DNAisolation method, DNA isolation, gap closure, and annotation of se-lected sequences; A.E., annotation of selected sequences and charac-terization of PMUs; S.A.M., project initiation, project support, andproviding materials and resources; A.J.R., sequencing and gap closure;D.V.S., construction of AY-WB genomic libraries, sequencing, andgap closure; K.T., bioinformatics (sequence assembly and gap closure);T.W., bioinformatics (maintenance of annotation database and auto-mated annotation); A.L., project manager for construction of AY-WBgenomic libraries, sequencing, and gap closure; J.W.C., project man-ager for bioinformatics (sequence assemply, gap closure, maintenanceof annotation database, and assembly); S.A.H., project initiation, over-all project management (experimental work, annotation, and all otherbioinformatics analyses), and writing of manuscript.
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inhabitants of humans, mammals, reptiles, fish, arthropods,and plants. Phytoplasmas are generally associated with arthro-pods and plants, whereas mycoplasmas (Entomoplasmatalesand Mycoplasmatales) and ureaplasmas (Mycoplasmatales) arepathogens that cause infections of the respiratory and urogen-ital tracts, eyes, alimentary canals, glands, and joints of humansand animals. Interestingly, three spiroplasmas, Spiroplasmakunkelii, Spiroplasma citri, and Spiroplasma phoeniceum, arealso insect-transmitted plant pathogens but belong to the orderEntomoplasmatales (34) and hence are distantly related to thephytoplasmas. Dual phytoplasma and spiroplasma infections ofinsects and plants occur frequently (40).
Severalmycoplasmas,ureaplasmas, spiroplasmas,andachole-plasmas have been cultured outside their hosts in artificialculture media. Culture media are complex, because mollicutessuffered extensive gene losses and consequently lack genes ofmany basic metabolic pathways. However, to date, phytoplas-mas have not been cultured in cell-free medium, indicatingthat phytoplasmas have a different metabolism and are likely tohave more highly reduced genomes than other mollicutes.
The aster yellows phytoplasma (AYP) strain witches’ broom(AY-WB) (“Ca. Phytoplasma asteris”; class Mollicutes) gener-ally spreads systemically in lettuce (Lactuca sativa L.) andChina aster (Callistephus chinensis Nees), inducing a variety ofsymptoms, including vein clearing, yellowing, stunting, witches’broom, pigment loss or sterility of flowers, and necrosis (99).The extreme malformations of plants suggest that phytoplas-mas interfere with plant hormone metabolism (51). AY-WBalso spreads systemically in Arabidopsis thaliana and Nicotianabenthamiana, inducing yellowing, stunting, and witches’ broomin both (X. Bai, V. Correa, and S. A. Hogenhout, unpublishedresults). AY-WB was classified into the 16SrI-A subgroup of“Ca. Phytoplasma asteris” based on the restriction fragmentlength polymorphism banding pattern of a 1.2-kb 16S rRNAgene PCR fragment (99). In contrast, onion yellows (OY)phytoplasma strain M (OY-M), the only other phytoplasma forwhich a complete genome sequence is available (74), belongsto the 16SrI-B subgroup (51). “Ca. Phytoplasma asteris,” pre-viously known as AYP or group I phytoplasma (52), is thelargest of the phytoplasmas and associates with more than 100economically important diseases worldwide (51, 62). Planthosts include broad-leaf, herbaceous plants and several woodyfruit crops (62).
AY-WB is transmitted by the polyphagous leafhopper Mac-rosteles quadrilineatus (Forbes). Phytoplasma interactions withinsects are complex and involve intra- and extracellular repli-cation in gut and salivary glands, epithelial and muscle tissues,and other organs and tissues. Whereas there is evidence thatsome phytoplasmas are vertically transmitted to the progeny oftheir insect vectors (37), the predominant means of survival ofphytoplasmas is through transmission between insects andplants. They appear to manipulate their insect and plant hoststo enhance their own transmission efficiencies. For example,AYPs can increase fecundity and longevity of their insect vec-tor, Macrosteles quadrilineatus (13).
Because of their small genomes and economic importance,mollicutes have been targeted for genome sequencing projectsfor some time. Mycoplasma genitalium was the second bacte-rium to be sequenced to completion because of its minimalgene complement for a cultivable organism (33). Thus far,
genomes of at least nine SEM clade mollicutes and one AAAclade mollicute (OY-M phytoplasma) (76) have been fullysequenced. Here, we report the full sequence of the smallgenome of AY-WB. Comparative genome analysis revealedthe presence of 14 to 23% repetitive DNA organized in po-tential mobile units (PMUs) in the phytoplasma genomes anddifferences in standard metabolic and nonmetabolic pathwaysbetween phytoplasmas and SEM clade mollicutes.
MATERIALS AND METHODSDNA isolation. The AY-WB strain was collected from diseased lettuce plants
in Celeryville, Ohio (41.00°N, 82.45°W), in 1998 (99). AY-WB was isolated fromlettuce plants about 2 weeks after the appearance of symptoms. The stems oflettuce plants were cut at several places with a sharp razor blade, and phloem sapoozing from the cut area was collected. On average, 1.6 ml sap was collected fromeach symptomatic lettuce plant. For preparation of gel plugs, 200 �l sap wasimmediately mixed with 800 �l precooled 30% glucose–1� Tris-EDTA (pH 8.0)buffer, followed by centrifugation at 16,000 � g for 20 min at 4°C. The pellet wasmixed with 80 �l 1% premelted low-melting agarose (45°C) in 0.5� Tris-borate-EDTA (pH 8.0) and incubated at 4°C. Solidified plugs were subjected to pro-teinase K digestion at 50°C for 48 h and then rinsed with 1� Tris-EDTA buffer(pH 8.0) three times before subjection to pulsed-field gel electrophoresis(PFGE). PFGE was conducted in a 1% agarose gel with a running time of 18 h,a 60- to 120-s switch time ramp, a voltage of 6 V/cm, and an included angle of120° (CHEF-DR III; Bio-Rad, Hercules, CA). The AY-WB chromosome pro-duced a single band of �700 kb in the PFGE gel. The identity of the band wasconfirmed by Southern blot hybridizations and PCR using phytoplasma-specificprobes and primers, respectively. The 700-kb fragment was excised from the gel,and the gel blocks were placed directly into the Elutrap (Schleicher & Schuell)collection chamber for elution of DNA at 106 V at 4°C for 15 h. DNA wasethanol precipitated using standard procedures and resuspended in deionizeddistilled water. The concentration of the purified genomic DNA was assessedusing a PicoGreen kit (Molecular Probes).
Sequencing strategy. The shotgun library was constructed at IntegratedGenomics Inc. (IG). Five micrograms of DNA was sheared using a computer-controlled shearing device (GeneMachines, San Carlos, CA) to produce DNAfragments of 2 kb on average. Sheared DNA was loaded onto 0.7% agarose gels,and DNA fractions corresponding to 2 to 2.5 kb were extracted from the agarosegel. Single-stranded ends of the DNA were removed by T4 polymerase and thenfilled in with Klenow fragment. Size-selected 2- to 2.5-kb DNA fragments werecloned into the pGEM-3Z vector (Promega, Madison, WI), introduced intoEscherichia coli DH10B, and sequenced with the DYEnamic ET Dye Terminatorkit (Amersham Biosciences, Piscataway, NJ). Sequence quality assessment andsubsequent assembly were performed with the Phred/Cross_match/Phrap pack-age (29, 30) and Paracel Genome Assembler. Sequencing and physical gaps inthe assembly were closed by multiplex PCR (92) and primer walking.
Annotation. The sequence data of AY-WB were submitted to the IG databaseand software suite, ERGO, for sequence annotation. CRITICA (8), Glimmer2(25), and IG proprietary tools were used for open reading frame (ORF) iden-tification. ORF function annotation was conducted by a number of IG propri-etary algorithms that automatically predict the function of ORFs based oncomparative analysis with orthologue clusters in ERGO. In addition, the pre-dicted proteins were searched, using the BLAST algorithm (6), against a non-redundant database at the National Center for Biotechnology Information(NCBI). Protein functional domains were analyzed by searching against theNCBI conserved-domain database (60) and the Pfam database (12). The Kyotoencyclopedia of genes and genomes was used for the reconstruction of themetabolic pathways. The assignment of Enzyme Commission (EC) number wasdone according to the BRENDA database (86).
Nucleotide sequence accession numbers. Sequences of the AY-WB genomehave been deposited in the GenBank database under accession numbersCP000061 (chromosome), CP000062 (plasmid AYWB-pI), CP000063 (plasmidAYWB-pII), CP000064 (plasmid AYWB-pIII), and CP000065 (AYWB-pIV).More detailed information on the AY-WB genome is available on our website(http://www.oardc.ohio-state.edu/phytoplasma).
RESULTS
General genomic features. The AY-WB genome is com-posed of one circular chromosome of 706,569 bp (Fig. 1A) andcontains two rRNA gene operons, 31 tRNA genes, and 671
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predicted ORFs (Table 1). UGA was used as a stop codon forthe prediction of the ORFs. This is consistent with other re-ports showing that acholeplasmas and phytoplasmas retainedUGA as a stop codon, unlike SEM branch mollicutes, whichuse UGA as a tryptophan codon instead of a stop codon (80).This is also in agreement with annotations conducted forOY-M (76). Our results were not in agreement with a previousreport that stated that UGA should be considered as a tryp-tophan codon in phytoplasmas, as in mycoplasmas (64). Theaverage guanine (G) and cytosine (C) content of the AY-WBchromosome is 27%. The genome has an irregular GC-skewpattern that is different from most prokaryotic genomes, whichusually consist of two major shifts near the origin of replicationand the terminus of replication (35). Irregular GC-skew pat-terns were also found in the genomes of some other bacteria,such as Wolbachia pipientis (97) and Mycoplasma mycoides(95). Because the location of the origin of replication (oriC)was not clear, the first nucleotide of the dnaA gene was as-signed as bp 1. However, oriC is most likely located upstream
FIG. 1. (A) Genome maps of the 706,569-bp circular chromosome of “Candidatus Phytoplasma asteris” strain AY-WB. Rings present from theinside to outside are as follows: ring 1, rrn operons in red and tRNA in green; ring 2, GC skew over a 2-kb window and 200-bp steps with reddenoting G � C and blue denoting C � G; ring 3, predicted ORFs in sense orientation in yellow and antisense orientation in blue; ring 4, locationof tra5 ISs presented as angular brackets with yellow indicating sense orientation and blue indicating antisense orientation; ring 5, ORFs presentin all sequenced mollicutes in blue and unique to phytoplasmas within the class Mollicutes in red; ring 6, ORFs of predicted secreted proteins ingreen, secreted membrane proteins in red, and membrane proteins in blue; ring 7, base pair indicator with the first nucleotide of dnaA as nucleotide1. oriC is most likely located immediately upstream of dnaA as predicted by Oriloc software (32) and by the opposite direction of ORFs surroundingthe putative oriC. (B) The four plasmids of AY-WB. ORFs are presented as block arrows with names of the ORFs on the outside of the rings.Numbers on the inside of the rings indicate the locations in base pairs, with the first nucleotide of the repA and rep genes as nucleotide 1. ORFsindicated with an asterisk are predicted to encode membrane-targeted proteins. In the GenBank database, the plasmids are referred to aspAYWB-I through pAYWB-IV. (C) Three chromosomal segments containing ORFs with similarity to plasmid ORFs. The chromosome ispresented as a black line. The numbers below the black lines indicate the positions of the first and last nucleotides of the sequence on the AY-WBchromosome in base pairs. ORFs are represented as block arrows. Arrows of paralogous genes on plasmids and chromosomes have the same color,with the exception of the gray arrows, which represent unique genes. The names of the ORFs with predicted functions are indicated above thearrows. RepA, plasmid replication-associated protein with significant similarity to RepA of geminiviruses. rep encodes the phytoplasma-specificplasmid replication protein encodes; ssb encodes the single-stranded DNA-binding protein.
TABLE 1. General features of the chromosomes ofAY-WB and OY-M
Feature AY-WB OY-Ma
Length (bp) 706,569 860,631G�C content (%) 27 28
Protein-coding region (%) 72 73No. of protein-coding genes with
assigned function450 446
No. of conserved hypothetical genes 149b 51No. of hypothetical genes 72 257
Total no. of genes 671 754
Avg length of protein-coding genes (bp) 779 785No. of tRNA genes 31c 32c
No. of rRNA operons 2 2
a Numbers taken from data reported previously by Oshima et al. (69).b Includes proteins with similarity (blastp, �10�5) to OY-M proteins.c tRNAs corresponding to all amino acids are represented.
3684 BAI ET AL. J. BACTERIOL.
of dnaA as predicted by Oriloc software (32) and by the op-posite direction of ORFs surrounding the putative oriC (Fig.1A) (35).
In addition to the chromosome, four small circular plasmidswere identified (Fig. 1B and Table 2). This was surprising,because the DNA isolation procedure should not allow theisolation of small DNAs. One explanation for this discrepancyis that the plasmids are present at high copy numbers in thephytoplasma cell. As a consequence, some plasmid DNA wascopurified from the PFGE gel along with the AY-WB chro-mosomal DNA. The plasmids contain a total of 22 putativeORFs, and their average GC contents ranged from 21.8% to25.6%. Each plasmid has genes for a replication initiationprotein (Rep) and a single-stranded DNA-binding protein(SSB) that are involved in rolling-circle amplification (45),whereas the functions of the other genes are not known. How-ever, most of the plasmid genes were predicted to encodesecreted or membrane proteins (Fig. 1B), and except for ORFpIII02 of AYWB-pIII and pIV06 of AYWB-pIV, all genes aresimilar to OY-M phytoplasma sequences (Table 2). It is strik-ing that whereas the plasmids encode different Rep proteins,they contain paralogous genes in similar orders (Fig. 1B). TwoAY-WB plasmids (AYWB-pI and AYWB-pIII) contain repAgenes similar to geminiviruses repA, whereas the rep genes ofthe other two plasmids (AYWB-pII and AYWB-pIV) wereunique to AY-WB and OY phytoplasmas.
The AY-WB plasmids seem prone to mutation. First, ORFspIII04 and pIII05 of AYWB-pIII are similar to the 5� and 3�portions, respectively, of paralogous genes on the other threeplasmids, suggesting that a mutation to a stop codon producedtwo ORFs in AYWB-pIII. Furthermore, the sequence betweenpII03 and ssb of AYWB-pII is similar to genes pI04, pIII06,and pIV04 of the other three plasmids but was not annotatedas an ORF because of the presence of a premature stop codon.In addition, plasmids apparently recombine with the chromo-some, as the latter contains three truncated ORFs similar tothe geminivirus-like repA plasmid genes and one truncatedcopy similar to the rep gene (Fig. 1C).
Repetitive and mobile DNA in the AY-WB genome. TheAY-WB genome contains long repeating units of DNA. Of the671 predicted ORFs of AY-WB, 191 (28%) ORFs, covering97,374 bp (13.8%) of the AY-WB chromosome, are present asmultiple copies (Fig. 2A). Of these 191 ORFs, 134 (20%),
covering 71,979 bp (10.2%) of the chromosome, are organizedas clusters, consisting of genes encoding transposases (tra5),DNA primases (dnaG), DNA helicases (dnaB), thymidylatekinases (tmk), Zn-dependent proteases (hflB), DNA-bindingproteins HU (himA), single-stranded DNA-binding proteins(ssb), and specialized sigma factors (sigF) and a number ofother genes with unknown function (Fig. 3). Many of thesehypothetical proteins are predicted to target phytoplasmamembranes (Fig. 1 and 3 and Table 3) and are therefore likelyinvolved in AY-WB interactions with plant and insect hosts.
The phytoplasma tra5 insertion sequences (ISs) belong tothe IS150 group of the IS3 family (53, 58). The presence of tra5ISs and other genes involved in recombination and repair, suchas himA, suggests that these cluster are mobile elements and,hence, were named PMUs. PMU1 is flanked by a complete tra5IS on one side and a truncated tra5 IS at the other side as wellas inverted repeats (IRs) of 327 bp (Fig. 3A). Sequences highlysimilar to the PMU1 inverted repeats were also found adjacentto the tra5 ISs of the other three PMUs (Fig. 3A). Anotherstriking observation is that all PMUs contain copies of dnaG,dnaB, ssb, and tmk that are involved in DNA replication, sug-gesting that the PMUs may transpose in a replicative fashion.
The AY-WB genome also contained several clusters thatlook like derivatives of PMUs, as they contained truncatedversions of PMU ORFs with gene orders similar to those ofPMUs. It is likely that these PMU-like clusters are in theprocess of being eliminated. Based on the positions of the tra5insertion sequences, the PMUs or PMU-like ORF clusters arepresent in at least seven locations in the AY-WB chromosome(Fig. 1A). At three locations in the AY-WB genome, PMUsare located adjacent to each other. The largest PMU-rich re-gion of the AY-WB chromosome is �75,000 bp (Fig. 1A),including PMU1 and PMU2 (Fig. 3A).
Not all dnaG, dnaB, tmk, hflB, himA, and ssb genes are partof PMUs or PMU-like clusters. As discussed above, several ssbgenes are located on plasmids or in plasmid-derived sequenceswithin the chromosome (Fig. 1B and C). The AY-WB chro-mosome also contains single copies of dnaG, dnaB, tmk, himA,and hflB homologs, which are clearly different in sequencefrom the PMU genes. Furthermore, AY-WB contains severalmulticopy sequences that are not part of PMUs, including onecomplete copy and several truncated copies of uvrD and dam.
TABLE 2. General features of the plasmids of AY-WB and OY-M
a Numbers taken from data reported previously by Oshima et al. (69).b Includes proteins with similarity (blastp, �10�5) to OY-M proteins.
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Comparative genome analysis of phytoplasmas. The AY-WBchromosome is 154,062 bp smaller than that of OY-M, andAY-WB has 83 fewer ORFs than OY-M (Table 1). This dif-ference in genome size is the result of a lower number ofmulticopy genes in AY-WB compared to OY-M (Fig. 2A).OY-M multicopy genes are also organized in PMUs. TheAY-WB genome contains 97,374-bp (13.8%; 191 ORFs) mul-ticopy sequences compared to 195,035-bp (22.7%; 268 ORFs)multicopy sequences for OY-M, and the majority are clusteredin PMUs with 71,979 bp (10.2%; 134 ORFs) for AY-WB and121,226 bp (14.1%; 175 ORFs) for OY-M. Thus, compared toOY-M, the 154,062-bp-smaller genome of AY-WB is due to97,661 bp fewer multicopy genes. The percentages of noncod-ing DNA are similar between AY-WB and OY-M, but becausethe OY-M genome is larger, OY-M noncoding DNA absorbsan additional 55,728-bp genome size difference between AY-WBand OY-M (Fig. 2A). As expected based on these observations,the numbers of single-copy ORFs are similar between the phy-toplasmas, with 432,553 bp (61.2%; 482 ORFs) for AY-WB and433,226 bp (50.3%; 486 ORFs) for OY-M (Fig. 2A).
The alignment of the AY-WB and OY-M genomes has anX-shaped pattern, illustrating synteny of the majority ofAY-WB and OY-M sequences but an inverse orientation oflarge genome segments (Fig. 2C). In both AY-WB and OY-M,the largest aligned region is �250 kb and starts with the lplAgene at 423,992 bp in AY-WB and 354,087 bp in OY-M andends with glnQ at 660,824 bp in AY-WB and 103,752 bp inOY-M (Fig. 2C, arrowheads). This region is upstream of theputative oriC in AY-WB but downstream of the putative oriCin OY-M. In both AY-WB and OY-M, these �250-kb regionscontain the majority of the metabolic genes and do not containtra5 insertion sequences (Fig. 1A).
The PMUs tend to congregate, as evidenced by the groups ofISs, and are frequently located on opposite strands, as can benoticed by the correlation of GC-skew inflection points and theboundaries of sense-antisense regions as well as tra5 insertionsequences in the AY-WB chromosome (Fig. 1A). The align-ment of the AY-WB and OY-M chromosomes revealed thatPMUs or PMU-like sequences at six locations in the AY-WBchromosome are also present at the same locations in theOY-M chromosome. However, at three locations, the se-quences in AY-WB or OY-M have undergone excessive dele-tion and mutation events. PMU sequences at one location inthe AY-WB chromosome and four locations in the OY-Mchromosome are unique to each of the phytoplasmas. Like
FIG. 2. Comparative analyses of the AY-WB genome with the ge-nomes of OY-M and other mollicutes. (A) The AY-WB and OY-Mgenomes are repeat rich. (B) Venn diagram showing the number ofshared and unique genes between AY-WB and OY-M. (C) Dot plotcomparison of AY-WB and OY-M chromosomes. The numbers on thex and y axes indicate the nucleotides in base pairs. AY-WB and OY-Mgenome segments in the same orientation are represented as red lines,
and those in the reverse orientation are represented as green lines. Thearrowheads indicate lplA and glnQ, which flank �250 kb of sequencesmostly conserved among mollicutes. (D) The number of ORFs uniqueto phytoplasmas or shared with sequenced SEM clade mollicutes basedon blastp analysis of AY-WB and OY-M protein sequences againsta database composed of deduced protein sequences of all fullysequenced mollicute genomes (E value, �10�5). GenBank acces-sion numbers are as follows: Mesoplasma florum L1, AE017263; My-coplasma gallisepticum R, AE015450; M. genitalium G-37, L43967;M. hyopneumoniae 232, AE017332; M. mobile 163K, AE017308; M.mycoides subsp. mycoides SC strain PG1, BX293980; Mycoplasmapenetrans HF-2, BA000026; Mycoplasma pneumoniae M129, U00089;M. pulmonis UAB CTIP, AL445566; OY-M phytoplasma, AP006628;Ureaplasma urealyticum serovar 3 strain ATCC 700970, AF222894.
3686 BAI ET AL. J. BACTERIOL.
AY-WB, the OY-M genome contains several genes that arenot part of PMUs, including two full-length and several trun-cated copies of dam and three full-length and several truncatedcopies of uvrD. Our observations are consistent with those ofothers, as Oshima et al. (76) previously reported that the OY-Mgenome contains multiple copies of uvrD, hflB, tmk, dam, andssb, constituting 18% of the total genes.
Besides the PMUs and other multicopy sequences, otherdifferences between AY-WB and OY-M were found. Strik-ingly, AY-WB lacks most sequences that are truncated inOY-M (Fig. 2B), including hsdR and hsdM of the type I re-striction modification system, three adjacent fragments withsimilarities to recA, and two adjacent sequences of the sucPgene for sucrose phosphorylase (EC 2.4.1.7). AY-WB alsolacks genes that are part of incomplete pathways in OY-M,including rfaG (EC 2.4.1.157) of the glycerolipid metabolismpathway and pdxK (EC 2.7.1.35) of the vitamin B6 pathway.Finally, whereas AY-WB lacks folC (EC 6.3.2.17) and hastruncated versions of folK (EC 2.7.6.3) and folP (EC 2.5.1.15),OY-M has full-length copies of these genes that belong to thefolate biosynthesis pathway. Only a few AY-WB ORFs withfunctional annotations were absent from OY-M (Fig. 2B).These include cbiQ and evbH of the cobalt and multidrugATP-binding cassette (ABC) transporter systems, respectively(Table 4). However, OY-M has chromosome fragments withsimilarities to cbiQ and evbH, but ORFs were not assigned.Except for these sequences, a high degree of gene contentconservation was observed between the genomes of AY-WBand OY-M, including major metabolic pathways and ABC andP-type ATPase transporters (76) (Tables 4 and 5).
Comparative genomics of phytoplasmas and other molli-cutes. To determine to what extent phytoplasma genomes dif-
fer from the distantly related SEM clade mollicutes, ORFsequences of the AY-WB and OY-M phytoplasmas were com-pared to those of nine Mycoplasma and Ureaplasma spp.(blastp; E value, �10�5). More than half of the phytoplasmaORFs had similarities to those of SEM clade mollicutes, andAY-WB and OY-M had an equal number of unique phyto-plasma ORFs (318 ORFs) (Fig. 2D). Relative to OY-M,AY-WB contained fewer ORFs that were present in severalbut not all SEM branch mollicutes (146 ORFs for AY-WBversus 214 ORFs for OY-M) (Fig. 2D). The �250-kb segmentbetween the lplA and glnQ genes that is syntenic between theAY-WB and OY-M phytoplasmas (Fig. 2C) contained themajority of the ORFs conserved among mollicutes (Fig. 1, bluepatches in ring 5), while the less syntenic region (the first 400kb of the AY-WB genome) (Fig. 2C) are repeat rich (Fig. 1 [ISelement ring 4] and 2C) and are more enriched with phyto-plasma-specific ORFs (Fig. 1, red patches of ring 5).
Of the 318 ORFs that are unique for phytoplasmas in theclass Mollicutes, 40 had functional annotations and were closelyexamined (Table 6), since these may be part of metabolicpathways absent from SEM branch mollicutes. These 40 ORFsinclude sfcA for NAD-specific malic enzyme (EC 1.1.1.38) andtwo copies of the malate/citrate-sodium symporter gene citS.Phytoplasmas have a maltose ABC transporter system, includ-ing a maltose-binding protein (MalE) (Table 4) and severalother transporters that are not present in the SEM clade mol-licutes (Table 6). These include several components of the artand gln ABC transporter systems that might be important forthe import of glutamine and arginine, respectively, and severalsolute-binding proteins, including ArtI, which is predicted tobind arginine (39); the dipeptide binding protein and D-ami-nopeptidase DppA (20); and NlpA lipoprotein (98), for which
FIG. 3. PMUs of the AY-WB chromosome. The chromosome is presented as a black line. The numbers between parentheses at the left indicatethe positions of the first and last nucleotides of the PMU on the AY-WB chromosome. ORFs are represented as block arrows. Arrows ofparalogous genes have the same color, with the exception of the gray arrows, which represent unique genes. The names of the ORFs with predictedfunctions are indicated above the arrows, with ORFs of predicted membrane-targeted proteins indicated with *. The ORF numbers below the arrowscorrespond to annotations listed in Table 3, with # indicating genes that contain mutations separating them into two truncated ORFs. However, the tra5ORFs of PMU4 contains separate A and B ORFs that may produce a full-length transposase upon a single frameshifting event (58).
VOL. 188, 2006 COMPARATIVE ANALYSIS OF TWO PHYTOPLASMA GENOMES 3687
the gene is located between methionine ABC transportergenes and which hence may be a methionine binding protein(Table 4). Phytoplasmas also have mntB and znuA of the man-ganese (Mn) and zinc (Zn) ABC transporter system (15) (Ta-ble 6). All the solute-binding proteins were predicted to havesignal peptides (SignalP v3.0) (14) and are likely extracellularlipoproteins (38). Two ABC transporters have adjacent genesfor thermostable carboxypeptidase 1 (EC 3.4.17.19) and oligo-endopeptidase F (EC 3.4.24.�) that can process imported pep-tides and that were not present in the genomes of SEM branchmollicutes (Table 6). Finally, three AY-WB genes were anno-tated as norM that encodes a Na�-driven multidrug effluxpump. One norM gene had similarity to genes of SEM molli-cutes, whereas the other two did not. These two are locatedadjacent to each other and are transcribed in opposite direc-tions in both the AY-WB and OY-M genomes.
Other genes present in AY-WB and OY-M but absent fromSEM branch mollicutes are pssA and psd (Table 6) of thephosphatidylethanolamine pathway (63). Furthermore, myco-plasmas lack pcnB encoding poly(A) polymerase (EC 2.7.7.19)
and pnp encoding polyribonucleotide nucleotidyltransferase(EC 2.7.7.8). Both are involved in the regulation of mRNAstability. Interestingly, the pnp gene is present in the genome ofS. kunkelii (9), which is also an insect-transmitted plant-patho-genic mollicute. Polyribonucleotide nucleotidyltransferase maybe involved in the persistent infection of insects and/or adap-tation to diverse hosts and habitats of phytoplasmas and spi-roplasmas (9). The adjoining phytoplasma genes pmbA andtldD were not identified in SEM branch mollicutes either.PmbA and TldD regulate DNA gyrase function and are in-volved in protein maturation (3, 70, 83).
Compared to other mollicutes, phytoplasmas lack severalessential transporters and pathways. AY-WB and OY-M lackphosphoenolpyruvate:sugar phosphotransferase (PTS) systemsfor the import of sugars essential for glycolysis. AY-WB andOY-M also lack F-type ATP synthases. This is in contrast tomycoplasmas and ureaplasmas that have ATP synthase com-plexes, including the A, B, and C subunits for the transmem-brane channel and the five-subunit (alpha, beta, gamma, delta,and epsilon) catalytic core for ATP synthesis, and can use the
TABLE 3. Features of the four PMUs of AY-WB
ORFaORF ID (length [nt])
Annotationg
PMU1 PMU2 PMU3 PMU4
1 tra5 (210)c Truncated transposase, groupIS150, family IS3
a ORF numbers correspond to numbers in Fig. 3.b Deduced proteins predicted to target the membrane (secreted or membrane proteins).c ORF IDs, with lengths in nucleotides shown in parentheses, are indicated for all PMU ORFs.d Genes contain mutations separating them into two truncated ORFs (Fig. 3).e Contains separate A and B ORFs that may produce a full-length transposase upon a single frameshift event (58).f Sequences unique to AY-WB.g Abbreviations: Cons, conserved; hyp, hypothetical.
3688 BAI ET AL. J. BACTERIOL.
TA
BL
E4.
AB
Ctransporter
genesin
AY
-WB
andO
Y-M
phytoplasma
genomes
SubstrateA
Y-W
BO
Y-M
AT
P-bindingprotein
Mem
braneprotein
Solute-bindingprotein
AT
P-bindingprotein
Mem
braneprotein
Solute-bindingprotein
Am
inoacid
uptakeA
mino
acidglnQ
(AY
WB
_634)A
YW
B635
(AY
WB
_635)glnQ
(39938565)artM
(39938563),artM(39938564)
D-methionine
metN
(AY
WB
_589)A
YW
B587
(AY
WB
_587)nlpA
(AY
WB
_588)abc
(39938618)PA
M134
(39938620)nlpA
(39938619)A
mino
acid(arginine)
AY
WB
_314(fragm
ent)glnP
(AY
WB
_315)artM
(39938942),artI(39938943),artM(39938950)
Am
inoacid
(glutamine)
artP(A
YW
B_264)
artQ(A
YW
B_265),
artM(A
YW
B_262)
artI(A
YW
B_263)
glnQ(39938974)
artM(39938973),artI
(39938975),artM(39938976)
Am
inoacid
artM(A
YW
B_125)
artM(39939074)
Am
inoacid
artM(39938980),artM
(39938981)A
mino
acidartM
(39939125),m
doB(39939127)
Dipeptide/oligopeptide
uptakeD
ipeptideor
oligopeptidedppF
(AY
WB
_527),dppD
(AY
WB
_528)dppB
(AY
WB
_530),dppC
(AY
WB
_531)dppA
(AY
WB
_529)dppD
(39938678)dppC
(39938675),dppB(39938676)
oppA(39938677)
Oligopeptide
dppD(39938511),
oppF(39938512)
dppB(39938508),
PAM
023(39938509)
PAM
024(39938510)
Sugaruptake
Maltose,trehalose,sucrose,orpalatinose
malK
(AY
WB
_670)m
alG(A
YW
B_668),
malF
(AY
WB
_669)m
alE(A
YW
B_667)
malK
(39939238)ugpE
(39939236),ugpA(39939237)
ugpB(39939235)
Inorganicion
uptakeC
obaltcbiO
(AY
WB
_014)cbiQ
(AY
WB
_015)cbiO
(39938506)PA
M19
(39938505)C
obaltcbiO
(AY
WB
_540),cbiO(A
YW
B_541)
cbiQ(A
YW
B_539)
cbiO(39938665)
cibQ(39938666)
Mn/Z
nm
ntA(A
YW
B_623)
mntB
(AY
WB
_622),m
ntB(A
YW
B_621)
znuA(A
YW
B_624)
znuC(39938579)
znuB(39938580)
znuA(39938578)
Multidrug
resistanceM
ultidrugevbG
/mdlB
(AY
WB
_028)m
dlB(39938545)
Multidrug
evbH(A
YW
B_029)
Spermidine/putrescine
uptakeSperm
idineor
putrescinepotA
(AY
WB
_095)potB
(AY
WB
_094),potC
(AY
WB
_093)potD
(AY
WB
_092)potA
(39939145)potB
(39939146),potC(39939147)
potD(39939148)
UncharacterizedL
ipoproteinphnL
(AY
WB
_619)phnL
(39938582)nlpA
(39938583)U
nknown
phnL(A
YW
B_135)
phnL(39939085)
VOL. 188, 2006 COMPARATIVE ANALYSIS OF TWO PHYTOPLASMA GENOMES 3689
transmembrane potential for ATP synthesis (80). However,phytoplasmas have five genes encoding P-type ATPases (Table5) that may generate electrochemical gradients over the mem-brane.
Phytoplasmas have fewer genes in the standard recombina-tion pathway and SOS response in comparison to SEM branch
mollicutes. All mollicutes sequenced so far lack recB, recC,recD, recG, and ruvC of the recombination pathway and recN,recO, recQ, and recR of the SOS response, although somemycoplasmas carry recR and recO. Thus, SEM branch molli-cutes have recA, recU, ssb, polA, gyrA, gyrB, ruvA, and ruvB, arudimentary set of genes that permit homologous recombina-
TABLE 5. Predicted P-type ATPases of AY-WB and OY-M
AYWB OY-M
Gene (length [aaa], ORF) Possible substrate Gene (length [aa],GenBank accession no.) Possible substrate
AYWB_561 hlyC a Hemolysin III 39938644 e�110 18145579 5e�26
AYWB_599 Immunodominant protein precursor 39938608 2e�10 NA NAAYWB_630 Rhodanese-related sulfurtransferase 39938571 e�180 23098027 e�109
AYWB_646 pduL PduL 39939215 e�102 49235943 5e�44
a Genes with identical annotations but no sequence similarities in SEM branch mollicute.b The E values were obtained by searching against the GenBank nonredundant database with 2,506,223 sequences consisting of 849,940,114 letters on a local Linux
workstation. Results from the GenBank search were verified using the mollicute database MolliGen (http://cbi.labri.fr/outils/molligen/) (11). NA, not available.
3690 BAI ET AL. J. BACTERIOL.
tion. Of these, phytoplasmas do not have recA, ruvA, and ruvB.Hence, phytoplasmas have a deficient homologous recombina-tion machinery.
AY-WB virulence. The AY-WB genome was analyzed forsimilarities to known bacterial virulence factors. Several puta-tive hemolysins of AY-WB were identified based on annota-tion. These include a protein annotated as HlyC, a putativehemolysin III. This protein belongs to the integral membraneprotein family (Pfam domain number PF03006), which in-cludes a protein with hemolytic activity from Bacillus cereus.However, other proteins in this family play a role in lipid andphosphate metabolic pathways. Another putative hemolysin-related protein of AY-WB was annotated as TlyC, which car-ries resemblance to cluster of orthologous group 1253 of he-molysins and related proteins containing CBS domains.Indeed, AY-WB TlyC contains a CBS domain (Pfam domainnumber PF00571). However, the AY-WB TlyC protein has anN-terminal transmembrane region (Pfam domain numberPF01595) not found in TlyC proteins and a C-terminal domainthat is present in the C terminus of Na�/H� antiporters, in-cluding CorC, which is involved in magnesium and cobalt efflux(Pfam domain number PF03471). Thus, it is not clear whetherHlyIII and TlyC of AY-WB are hemolysins.
Two AY-WB proteins, AYWB_084 and AYWB_352, aresimilar to the Legionella pneumophila virulence factor IcmE (Evalues of 5e�21 and 5e�05, respectively), which is part of thetype IVB secretion system apparatus that translocates bacterialproteins into host cells (87). Proteins with similarities to IcmEwere also identified in the OY-M genome (76). IcmE hassequence similarity to plasmid genes involved in conjugation(87). In both AY-WB and OY-M, the majority of the icmE-likesequences were located upstream of the ATP-dependent heli-case gene uvrD. UvrD belongs to the Rep family of helicasesand catalyzes ATP-dependent mediated unwinding of double-stranded DNA into single-stranded DNA and has a role in therecF recombination pathway, methyl-directed mismatch repair,and UvrABC-mediated nucleotide excision repair and replica-tion (36, 67). Similarly to the other repeated sequences, theOY phytoplasma genome contains multiple copies of icmE-like sequences and full-length uvrD, whereas the AY-WB phy-toplasma contains only one full-length icmE-like sequence anduvrD and multiple truncated copies of these sequences. Fur-ther research should reveal whether the icmE-like sequencesof phytoplasmas mediate conjugation or are somehow involvedin the recombination pathway. No other similarities of phyto-plasma sequences to type III and type IV secretion systemswere observed. This may not be surprising, as translocation ofvirulence factors via type III and type IV secretion systems ismore specific for gram-negative bacteria.
AY-WB and OY-M share the genes of the protein exportand targeting components of the sec-dependent pathway, in-cluding secA, secY, yidC, ffh, ftsY, dnaJ, dnaK, grpE, groES, andgroEL and, like SEM branch mollicutes, lack several subunitsand the signal peptidases of the protein maturation compo-nent, including secB, secG, secF, secE, secD, and signal pepti-dase I (80). Despite the absence of several components, OY-Mphytoplasma has a functional sec-dependent protein transloca-tion system (43). It is possible that some of the many hypo-thetical proteins have peptidase activities. This confirms pre-vious findings (10, 44) that phytoplasmas have a functional
sec-dependent protein translocation system and that the N-terminal signal peptides of proteins are cleaved. Since theclosest walled relatives of phytoplasmas are Clostridium, Bacil-lus, and Streptococcus spp. (phylum Firmicutes), it is possiblethat, similarly to Streptococcus pyogenes (84), phytoplasmas se-crete virulence-related proteins via the sec-dependent pathway.
Both phytoplasma genomes contain several ABC transport-ers (Table 4). ABC transporters import peptides, amino acids,and nutrients into the cell. They can be virulence factors, andthey can deplete essential nutrients from the host and secretetoxins and antimicrobial compounds such as hemolysins (23).Furthermore, solute-binding proteins of ABC transporters areusually secreted lipoproteins that bind external substrate to thecell and deliver the substrate to the ABC transporters and mayalso be involved in adherence to cell surfaces (4). For instance,the ABC transporter-related solute-binding protein Sc76 ofSpiroplasma citri was shown to be involved in the penetrationof or multiplication in the salivary gland (17). The AY-WBgenome contains genes for five solute-binding proteins withspecific solute-binding activities (Table 4). All five solute-bind-ing proteins have N-terminal cleavable signal peptide se-quences, as predicted with SignalP v3 software (14), and there-fore are secreted via the sec-dependent pathway. Hence, thesefive solute-binding proteins are putative virulence factors ofphytoplasmas.
DISCUSSION
It is intriguing that phytoplasmas have small genomes thatlack many standard metabolic functions but are repeat rich.The repeated DNAs are mostly multicopy genes organized inPMUs. Thus, phytoplasmas are different from other bacterialendosymbionts of insects, e.g., Buchnera and Blochmanniaspp., which also have small genomes lacking many standardmetabolic functions but have low levels of repeated DNAs (1,91). On the other hand, the majority of the mollicutes haverepeat-rich genomes. All mollicutes are under pressure forgenome minimization, and the presence of numerous repeatsis therefore highly significant (82). Indeed, it has been shownfor several mycoplasmas that repeats engage in recombinationevents resulting in changes of mosaics of antigenic structures atcell surfaces, essential for evasion of the host immune systemand for adaptation to new environments (82). Thus, similarlyto mycoplasmas, the repeated DNAs of phytoplasmas probablyallow adaptations to different environments. Adaptation is par-ticularly important for phytoplasmas, as their host environ-ments are extremely variable, including the intracellular envi-ronments of phloem tissues of plants and guts, salivary glands,and other organs and tissues of insect hosts. Also, phytoplas-mas have a broad plant host range. AY-WB alone can infectChina aster, lettuce, tomato, Nicotiana benthamiana, and Ara-bidopsis thaliana. Phytoplasma genomes are different from my-coplasma genomes in several aspects. First, phytoplasmas donot have recA, ruvA, and ruvB and hence appear to lack afunctional recombination system. Second, thus far, the organi-zation of repeated DNAs in PMUs (Fig. 3 and Table 3) isunique to phytoplasmas among the mollicutes.
PMUs. The PMUs contain tra5 ISs, which belong to theIS150 group and the IS3 family (53, 58). IS3-type mobile unitsare found in a number of other mollicutes, for example, IS1138
VOL. 188, 2006 COMPARATIVE ANALYSIS OF TWO PHYTOPLASMA GENOMES 3691
in Mycoplasma pulmonis, IS1221 in Mycoplasma hyorhinis andMycoplasma hyopneumoniae, IS1297 in M. mycoides subsp. my-coides, ISMi1 in Mycoplasma fermentans, and one IS3 elementin the spiroplasma virus DNA SPV1-C74 sequence of S. citri(58, 66). All of these elements belong to the IS150 subgroup,and it has been demonstrated that some of these elementsundergo autonomous transposition (16).
PMU1 of AY-WB is the longest, appears to be the mostcomplete, and has several striking features characteristic ofcomposite transposons (Fig. 3). First, the right and left bordersof PMU1 contain long (327-bp) IRs. Furthermore, whereas theORF to the right is a truncated tra5 sequence, the tra5 se-quence at the left can produce a full-length ORFAB fused-frame transposase (58). IS150 can generate circles by joiningIRs upon production of the fused-frame transposase (90), andparticularly, composite transposons that carry single invertedrepeats at the left and right borders form stable circles (48).PMU1 also carries a gene for DNA protein HU (himA), whichis a nonspecific binder of DNA but prefers binding to bent,kinked, or altered DNA sequences (31) and has a role inrecombination through the joining of distant recombinationsites (5). Thus, with the help of transposase and DNA proteinHU, the IRs could join to form a circle and induce transposi-tion of PMU1. It is striking that all the genes on PMUs areoriented in the same direction, with sigF, encoding a special-ized transcription factor, as the first gene and located down-stream of the inverted repeat. In IS3 family members, theadjoined IRs, which are formed on circularization, create astrong hybrid promoter that drives high levels of transposaseexpression (58). Hence, it is possible the adjoined 327-bp re-peats upon circulation of PMU1 create a strong promoter thatdrives the transcription of at least part of the PMU genes.
The AY-WB and OY-M genomes also contain evidence thatat least some PMUs transpose in a replicative fashion. First,there are multiple copies of PMUs and PMU-like clusters.Second, the PMUs contain full-length dnaB, dnaG, and ssbgenes that are involved in DNA replication. DnaB initiatesDNA replication (19). It moves along the lagging strand andunwinds the DNA helix for the propagating fork and attractsDnaG for lagging-strand synthesis (93). SSB plays an essentialrole in DNA replication by stabilizing single-stranded DNA(56). Most PMUs also contain a tmk gene encoding thymidy-late kinase that synthesizes dTDP from dTMP for DNA syn-thesis. Similarly to AY-WB, the OY-M phytoplasma genomecontains at least two tmk homologs, tmk-a and tmk-b, withtmk-a being present as multiple copies (68). We revealed thatthe tmk-a genes are part of PMUs. However TMK-b but notTMK-a was shown to have thymidylate kinase activity (68).Hence, the function of TMK-a is not yet clear.
Several sigma factor genes were identified in the AY-WBgenome. These genes are rpoD, which encodes the standard465-amino-acid 70 protein and is present as a single copy onthe AY-WB chromosome, and multiple copies of sigF that arelocated on PMUs or PMU-like gene clusters and have deducedproteins of �200 amino acids in length. PMU3 contains asequence with similarity to sigF immediately upstream of thessb gene, but because of the presence of a premature stopcodon, this sequence was not predicted to be an ORF. TheOY-M genome also has multiple copies of sigF that are part ofPMUs. The N-terminal 100 amino acids of the SigF proteins
have region 2 domains (Pfam domain number PF04542) con-taining both the �10 promoter recognition helix and the pri-mary core RNA polymerase binding determinant. However,the C-terminal 100 amino acids of the SigF proteins do nothave similarities to other proteins or domains, including theregion 4 domains (Pfam domain number PF04545) containingthe �35 promoter-binding element. AY-WB SigF proteinsshowed the greatest similarity (E value, 10�6) to the stressresponse sigma factor [sigma(H)] of Streptococcus coelicolor(50) and the flagellar biosynthesis sigma factor FliA of Pseudo-monas putida (46). Expression of SigF and other PMU genesmight occur under specific environmental conditions.
Since PMUs contain several genes predicted to encodemembrane-targeted sequences, one would expect that expres-sion of PMU genes would result in a change of the phyto-plasma membrane surface. In this regard, it is intriguing thatthe PMUs contain hflB (or ftsH) genes that encode membrane-associated ATP-dependent Zn proteases of �700 amino acids.These proteins are conserved among bacteria and are involvedin membrane-associated processes such as protein secretion(26) and membrane protein assembly (2) as well as adaptationsto nutritional conditions and osmotic stress (26, 57).
Genomic plasticity. The irregular GC skews and the pres-ence of large repeated sequences (PMUs) in the AY-WB andOY-M genomes are indicative of high genomic plasticity. Thecorrelation between an irregular GC skew and the presence ofISs in mollicute genomes is quite striking. For instance, M.mycoides has an irregular GC skew, and 13% of the genomesize consists of ISs (95), whereas Mycoplasma mobile has aregular GC skew and no ISs (42). It should be noted, however,that although AY-WB doesn’t have a significant consistent GCskew, it may have another kind of significant skew or excess,including AT skew and purine excess or keto excess (89).
Phytoplasma genomic plasticity is also evidenced by the dif-ferences in genome sizes and compositions between membersof “Ca. Phytoplasma asteris,” ranging from 660 to 1,130 kb andconsisting of several fragments of 500 kb and larger (61; ourpersonal observation). Since PMUs can form large clustersthat may locate in different sections of the chromosome, it islikely that they are also capable of splitting a single chromo-some into two smaller chromosomes. Furthermore, results re-ported herein show that AY-WB and OY-M differ by �154 kbin genome size, mainly because of a difference in PMUs andother multicopy sequences (Fig. 2A).
Despite the phytoplasma genomic plasticity, the majority ofthe AY-WB and OY-M genomes are syntenic (Fig. 3C). Scat-ter plots of conserved sequences between the AY-WB andOY-M genomes show an X-shaped pattern with symmetryaround the tentative oriC and two other locations at approxi-mately opposite ends of oriC (Fig. 2C). This X-shaped patternor X alignment is common in genome comparisons of closelyrelated bacterial species and is most likely due to the occur-rence of large inversions that rotate around oriC and the ter-minus of replication (28). The breakpoints of the inversionsbetween the AY-WB and OY-M genomes are, as expected, atPMU-like regions and repeated uvrD sequences.
There are probably two reasons for the good alignment ofthe AY-WB and OY-M genomes. First, we already observedthat the PMUs tend to congregate. This is consistent withfindings that IS150 frequently transposes into target regions
3692 BAI ET AL. J. BACTERIOL.
resembling its IR (58, 74). Thus, transposition will predomi-nantly affect certain areas of the phytoplasma genomes, andhence, the synteny in the rest of the genome can be main-tained. Second, because of the absence of recA, ruvA, and ruvB,rearrangements between PMUs through homologous recom-bination are likely to occur at lower frequencies than in ge-nomes with RecA-dependent homologous recombination ma-chineries (78, 79).
Variations in the presence of recA are common among in-sect-associated mollicutes (65). Truncated recA genes werefound in six Spiroplasma citri strains, which, like phytoplasmas,are insect-transmitted plant pathogens, and five Spiroplasmamelliferum strains, which are pathogens of bees (59). In S. citri,only the first 390 nucleotides at the 5� end of recA are present,whereas in S. melliferum, the full-length recA gene is inter-rupted by a TAA stop codon. Intriguingly, truncated and full-length RecA polypeptides were observed in a proteomic studyof S. melliferum (21). These finding suggest that recA sequencevariation among insect-associated mollicutes is of biologicalsignificance. RecA has an important function in mycoplasmas.Deletion of recA is lethal for M. pulmonis (80). RecA is prob-ably essential for homologous recombination between re-peated lipoproteins, and adhesin genes result in a change ofmosaics of antigenic structures at the bacterial surface, withsubsequent evasion of the host immune response (80, 82).Thus, it seems that phytoplasmas and spiroplasmas can adaptto their hosts with less efficient homologous recombinationsystems, and the loss of RecA function might then be beneficialfor increasing genome stability. This is supported by the ob-servations that, like phytoplasmas, spiroplasmas have highlyrepeat-rich genomes mainly due to phage-derived sequences(80). On the other hand, M. mycoides, which also has a repeat-rich genome and is a human pathogen, has a full-length recA (47).
Reductive evolution. In general, AY-WB seems furtheralong in the reductive evolution process than OY-M. First,AY-WB phytoplasma contained fewer PMUs insertions, andthe ORFs in AY-WB PMUs are more frequently truncated ordeleted. Second, AY-WB lacks genes that are truncated inOY-M, including asnB, hsdR, hsdM, recA, and sucP. Third,AY-WB lacks genes of incomplete pathways in OY-M, includ-ing rfaG of the glycerolipid metabolism pathway and pdxK ofthe vitamin B6 pathways. Furthermore, unlike OY-M, AY-WBdoes not have folC, and OY-M has full-length folK and folPgenes that are truncated in AY-WB. The folK and folP geneswere also identified as pseudogenes in clover phyllody (CPh)phytoplasma (“Ca. Phytoplasma asteris”) (24), suggesting thatOY-M may be capable of de novo folate synthesis, whereasAY-WB and CPh have to import folate from host cells. Simi-larly to CPh (24), the folK and folP sequences of AY-WB andOY-M are flanked by gcp, which encodes a glycoprotease, andtwo ORFs encoding a DegV family protein and a 24-kDalipoprotein (AYWB_245) (24). Hence, the gene organizationsof this part of the genome are conserved among “Ca. Phyto-plasma asteris” members. Final evidence that AY-WB is fur-ther down the reductive evolutionary path is provided by theobservation that relative to OY-M, AY-WB contains fewerORFs that are shared by several but not all mollicutes (146ORFs for AY-WB versus 214 ORFs for OY-M) (Fig. 2D).
Plasmids. We identified four plasmids in AY-WB. Plasmidshave been detected in a number of other phytoplasmas (55,
73). Each AY-WB plasmid contains two genes involved inrolling-circle amplification and two to six ORFs with unknownfunction, several of which were predicted to target the AY-WBmembrane, suggesting that the plasmids are involved inAY-WB association with the plant and insect hosts. Indeed,the RepA proteins of OY-M phytoplasmas were detected ininfected plants (71), indicating that the plasmid genes areexpressed during infection of the plant. Furthermore, sponta-neous OY-M mutants that lack ORFs on a plasmid and are notinsect transmissible were isolated (72).
Interestingly, two AY-WB plasmids (AYWB-pI andAYWB-pIII) contain repA genes similar to geminivirus repA,whereas the rep genes of the other plasmids were unique toAY-WB and OY-M phytoplasmas. Geminivirus-like repAgenes in OY-M (75) and more distantly related phytoplasmas(55, 81) were also identified. Like phytoplasmas, geminivirusesare insect-transmitted plant pathogens and have to passthrough the gut epithelium, hemolymph, and salivary glandcells of the insect vectors before returning to the plant (22).Phytoplasmas and geminiviruses have overlapping plant andinsect host ranges. Hence, it is possible that phytoplasmasacquired the repA genes from geminiviruses through horizontalexchange. On the other hand, it has been hypothesized thatgeminiviruses originated from bacterial plasmids (49). Plas-mids with similar repA genes are generally incompatible, andtherefore, it is likely that the four plasmids are not present inone AY-WB cell but represent the plasmid content of theAY-WB population present in plants from which the AY-WBDNA was isolated.
The variation among the AY-WB plasmids suggests thatthey are prone to frequent mutations. This is consistent withother findings. OY-M has plasmids ranging from �3 to �7 kbin size (Fig. 1B) (73), and the plasmids of beet leafhopper-transmitted virescence phytoplasma range from �2.5 to �11kb (55). There is high variability of the occurrence of ORFs inthe plasmids of 30 beet leafhopper-transmitted virescence phy-toplasma strains (55). There is also evidence of intramolecularrecombination among phytoplasma plasmids (55, 73). Weshow that they can also recombine with the chromosome (Fig.1C).
Phytoplasma metabolism. Except for a few exceptions de-scribed above, the AY-WB metabolic pathways are similar tothose of OY-M that have been described elsewhere (76) andwill not be discussed in detail here, although a few findingsneed more emphasis. The phytoplasma metabolism is in sev-eral ways different from those of SEM branch mollicutes. Thiswas expected, because phytoplasmas have not yet been grownin cell-free culture media, including mycoplasma culture me-dia. Unlike SEM branch mollicutes, phytoplasmas do not havePTS systems to import sugars and to generate glucose-6-phos-phate to feed the glycolysis pathway. Thus, phytoplasmas areclearly different from the insect-transmitted plant-pathogenicS. citri and S. kunkelii, which have three PTS systems for theimport of fructose, glucose, and trehalose (7). In contrast,phytoplasmas possess ABC transporters for the import of mal-tose. The maltose-binding protein (MalE) (Table 4) may haveaffinity to maltose, trehalose, sucrose, and palatinose (88). Af-finity of MalE to trehalose is likely, as trehalose is a majorsugar in the insect hemolymph. The fate of these sugars afterimport is not clear, because enzymes required for the conver-
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sion of these sugars to glucose-6-phosphate for glycolysis werenot found in the phytoplasma genomes, and the sucrose phos-phorylase gene, which is important for sucrose degradation, isfragmented in the OY-M phytoplasma genome (76) and iscompletely absent from the AY-WB phytoplasma genome (Ta-ble 6). Generally, the genomes of AY-WB and OY-M phyto-plasmas harbor significantly fewer carbohydrate transport andmetabolism genes than their mycoplasma counterparts. Evenin the 580-kb genome of M. genitalium, 26 carbohydrate trans-port and metabolism genes were identified (33). In contrast,only 19 genes are present in the 860-kb OY-M phytoplasmagenome (76), and 16 genes are present in the 706-kb AY-WBphytoplasma genome.
Unlike SEM branch mollicutes, phytoplasmas have a NAD-specific malic enzyme (EC 1.1.1.38) and malate/citrate-sodiumsymporter genes. Thus, like symbiotic Rhizobium spp. (77) butunlike sequenced SEM branch mollicutes, phytoplasmas may usemalate as a carbon source. The use of malate is advantageous,because it is readily available in the cytoplasm of host cells, andit can serve as the sole energy source for bacteria by conversionto oxaloacetate and pyruvate (27, 77). Furthermore, metabo-lism of malate saves energy (27), which is important, becausephytoplasmas lack ATP synthases, and hence, the capacity togenerate energy in phytoplasmas seems limited to glycolysis(starting with glucose-6-phosphate).
Unlike SEM clade mollicutes, phytoplasmas appear to becapable of biosynthesis of their own membrane phospholipids.The genomes of AY-WB, OY-M (76), and Western X-diseasephytoplasma (54) contain the pssA and psd genes (Table 6)encoding CDP-diacylglycerol-serine-O-phosphatidyltransferase (EC2.7.8.8) and phosphatidylserine decarboxylase (EC 4.1.1.65),respectively. Both are part of the phosphatidylethanolaminepathway (63). Furthermore, the AY-WB and OY-M genomescontain a candidate pmt gene for phospholipid N-methyltrans-ferase (Table 6) that is involved in phosphatidylcholine syn-thesis in conjunction with PssA and Psd (63). This confirmsthat phytoplasmas are phylogenetically more related to acho-leplasmas (4), which do not require exogenous phospholipids,whereas SEM branch mollicutes are sterol and fatty acid auxo-trophs (80). AY-WB and OY-M also have all enzymes that linkthe glycolysis pathway to the glycerolipid pathway (76) and anABC transporter gene, phnL, involved in lipoprotein release(Table 4).
Summary. Phytoplasmas have intriguing genomes that aresmall and contain many multicopy sequences mainly organizedas PMUs. The AY-WB genome is �154 kb smaller than theOY-M genome, primarily as a result of fewer multicopy se-quences. Thus, expansions or reductions of PMUs play a majorrole in phytoplasma genome evolution. At least one PMU,PMU1, has the characteristics of a replicative composite trans-poson. PMUs contain genes for specialized sigma factors andmembrane proteins, providing evidence that PMUs are impor-tant for phytoplasma interactions with the environment. Sincephytoplasmas lack recA and other standard homologous re-combination functions, it is unlikely that phytoplasmas gener-ate antigenic variation of membrane proteins through RecA-dependent homologous recombination. We propose that theregulation of expression of PMU genes is one of the strategiesphytoplasmas use to adapt to different environments. Expres-sion of PMU genes might occur through a process that involves
circularization and replicative transposition. In addition, ge-nome rearrangements through expansions and deletions ofPMUs might increase the chance of phytoplasma adaptation todiverse hosts and can be a major evolutionary factor allowingphytoplasmas to occupy broad plant host ranges or to adapt todifferent insect vectors. Few genes have similarities to knownbacterial virulence factors. Like the related gram-positive bac-teria, phytoplasmas may secrete virulence-related proteins viathe Sec-dependent pathway. Hence, all the proteins with signalpeptides are potential virulence factors, including the five sol-ute-binding proteins of the ABC transporters and proteinsderived from plasmids and PMUs. Finally, phytoplasmas haveABC transporters for the import of maltose (or trehalose,sucrose, and palatinose), utilize malate, and can make phos-pholipids. In contrast, SEM branch mollicutes have PTSs forthe import of fructose, glucose, and trehalose, utilize lactate,and are phospholipid auxotrophs.
ACKNOWLEDGMENTS
This work was supported by the National Research Institute of theUSDA Cooperative State Research, Education, and Extension Ser-vice, grant 2002-35600-12752, and the Ohio Agricultural Research andDevelopment Center competitive grants program.
We thank former members of the bioinformatics and genome anal-ysis group at Integrated Genomics, including Svetlana Gerdes, EugeneGoltsman, Viktor Joukov, Vinayak Kapatral, Yakov Kogan, NikosKyrpides, Andrei Osterman, Olga Ostrovskaya, and Ross Overbeek.We also acknowledge Angela D. Strock, Melanie L. Lewis Ivey, andJhony Mera for excellent technical assistance.
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