‘Ca. Liberibacter asiaticus’ Proteins Orthologous with pSymA-Encoded Proteins of Sinorhizobium meliloti: Hypothetical Roles in Plant Host Interaction

Sinorhizobium meliloti strain 1021, a nitrogen-fixing, root-nodulating bacterial microsymbiont of alfalfa, has a 3.5 Mbp circular chromosome and two megaplasmids including 1.3 Mbp pSymA carrying nonessential ‘accessory’ genes for nitrogen fixation (nif), nodulation and host specificity (nod). A related bacterium, psyllid-vectored ‘Ca. Liberibacter asiaticus,’ is an obligate phytopathogen with a reduced genome that was previously analyzed for genes orthologous to genes on the S. meliloti circular chromosome. In general, proteins encoded by pSymA genes are more similar in sequence alignment to those encoded by S. meliloti chromosomal orthologs than to orthologous proteins encoded by genes carried on the ‘Ca. Liberibacter asiaticus’ genome. Only two ‘Ca. Liberibacter asiaticus’ proteins were identified as having orthologous proteins encoded on pSymA but not also encoded on the chromosome of S. meliloti. These two orthologous gene pairs encode a Na+/K+ antiporter (shared with intracellular pathogens of the family Bartonellacea) and a Co++, Zn++ and Cd++ cation efflux protein that is shared with the phytopathogen Agrobacterium. Another shared protein, a redox-regulated K+ efflux pump may regulate cytoplasmic pH and homeostasis. The pSymA and ‘Ca. Liberibacter asiaticus’ orthologs of the latter protein are more highly similar in amino acid alignment compared with the alignment of the pSymA-encoded protein with its S. meliloti chromosomal homolog. About 182 pSymA encoded proteins have sequence similarity (≤E-10) with ‘Ca. Liberibacter asiaticus’ proteins, often present as multiple orthologs of single ‘Ca. Liberibacter asiaticus’ proteins. These proteins are involved with amino acid uptake, cell surface structure, chaperonins, electron transport, export of bioactive molecules, cellular homeostasis, regulation of gene expression, signal transduction and synthesis of amino acids and metabolic cofactors. The presence of multiple orthologs defies mutational analysis and is consistent with the hypothesis that these proteins may be of particular importance in host/microbe interaction and their duplication likely facilitates their ongoing evolution.


Introduction
Within the order Rhizobiales of the class alpha-Proteobacteria, Sinorhizobium meliloti is a member of a diverse bacterial family, Rhizobiaceae [1][2][3]. Other genera contained within the Rhizobiaceae and indeed the Rhizobium type genus [3] include Agrobacterium and Rhizobium [4]. A growing number of species of the family Rhizobiaceae and of the order Rhizobiales are becoming recognized based on 16 S RNA gene sequence data. Many species interact with plants and other eukaryotes to produce important agricultural, economic and/or environmental consequences. 'Ca. Liberibacter asiaticus' is recognized as the causal agent of huanglongbing (HLB), also known as citrus greening disease. This phytopathogen was introduced into the New World from Asia [5,6], and is transmitted by the citrus psyllid, Diaphorina citri [7]. The full genomic sequence of 'Ca. Liberibacter asiaticus' was determined by deep sequencing of total DNA obtained from a single citrus psyllid which contained more than 10 8 cells of 'Ca. Liberibacter asiaticus.' Phylogenetic analysis indicated that 'Ca. Liberibacter asiaticus' and S. meliloti are close bacterial relatives [2]. 'Ca. Liberibacter asiaticus' has a single small chromosome of 1.23 Mbps with 36.5% GC content compared to the chromosome of S. meliloti which is 3.65 Mbps and 62.7% GC [2,8]. 'Ca. Liberibacter asiaticus' is not available in culture but can colonize intracellularly and move systemically in the phloem vessels of citrus trees [9,10] following introduction by the citrus psyllid, D. citri. 'Ca. Liberibacter asiaticus' also systemically and intracellularly colonizes citrus psyllid salivary glands [11]. The genome of 'Ca. Liberibacter asiaticus' has many adaptations to facilitate an intracellular lifestyle in both plant and insect cells [2,12].
S. meliloti 1021, free living in the soil, also lives in specialized root nodules in symbiosis with alfalfa, Medicago sativa L., where S. meliloti reduces atmospheric nitrogen and thereby confers plant nitrogen sufficiency. S. meliloti 1021 has a composite genome comprised of the chromosome and two megaplasmids, pSymA and pSymB, of 1.3 Mbps and 1.7 Mbps [8]. Megaplasmids pSymA and pSymB encode 1293 and 1570 protein-coding genes, respectively [8,13].
Predicted gene products of all open-reading frames (ORFs) in the genome of 'Ca. Liberibacter asiaticus' were compared by protein BLAST against all of the predicted gene products encoded by chromosomal genes of S. meliloti 1021 [2]. However, predicted gene products of all ORFs encoded on the plant symbiosiscontrolling S. meliloti megaplasmid pSymA had not been previously subjected to protein BLAST compared with the predicted gene products encoded in the 'Ca. Liberibacter asiaticus' genome. As an ''accessory'' genome component [14], pSymA may have originated from an ancestral plasmid and may have maintained individual and some small blocks of genes originally from host chromosomes. It has long been known that the pSymA megaplasmid of S. meliloti, in contrast to the circular chromosome or the other S. meliloti megaplasmid pSymB, specializes in carrying genes essential for establishing and maintaining intimate intracellular plant interactions with alfalfa. The megaplasmid pSymA itself is self-transmissible and upon transfer confers nodulation (nod), nitrogen fixation (nif) and other symbiotic abilities in related bacteria with a similar genetic background or complement of chromosomal genes [15][16][17]. The chromosome of the tumor-inducing bacterial phytopathogen Agrobacterium tumefaciens encodes characteristics practically identical to those of Rhizobium microsymbionts, and members of the species also maintain pTi as an accessory genomic component. When bacterial cells recruit or accept transfer of the Ti plasmid or pSymA, either tumor-inducing pathogenicity or nitrogen-fixing symbiotic ability, respectively, is conferred on the host bacterium. Agrobacterium and Rhizobium were combined into a single genus within the Rhizobiaceae largely for this reason and to reject taxonomy based largely on plant interactions determined by extrachromosomal elements, i.e., to avoid plasmid-based nomenclature [4].
As S. meliloti is a close phylogenetic relative of 'Ca. Liberibacter asiaticus,' it is likely that the two bacteria deploy a similar repertoire of mechanisms for avoiding defenses elicited in host plant cells by their invasion, or, in the case of beneficial root nodule bacteria, recruitment or ''welcomed entry'' [18]. The goal of the present study was to compare predicted gene products encoded by the genome of 'Ca. Liberibacter asiaticus' with those encoded by genes carried on the megaplasmid pSymA of S. meliloti strain 1021. The rationale for identifying protein products of 'Ca. Liberibacter asiaticus' genes orthologous to those encoded by genes on pSymA is simply that such genes and their protein products may play direct or indirect role(s) in establishing and maintaining an intimate intracellular interaction with eukaryotes. We have identified a number of such interesting protein products.

Maintenance of Homeostasis
In general, proteins encoded by pSymA genes are more similar in sequence alignment to those encoded by S. meliloti chromosomal orthologs than to orthologous proteins encoded by genes carried on the 'Ca. Liberibacter asiaticus' genome. There are two very notable exceptions where orthologs are shared only by pSymA and 'Ca. Liberibacter asiaticus' and not with the chromosome of S. meliloti. YP_003064907, the predicted protein product of a 'Ca. Liberibacter asiaticus' gene, is orthologous to NP_435608.2 (E = 5e-71) encoded by pSymA yet lacks an orthologous protein encoded by the chromosome of S. meliloti (Table 1). SMART analysis predicts that YP_003064907, has a cation efflux domain (E = 1.3e-31) from residues 15 to 313 of the 313 amino acid protein. This predicted protein also has BLAST matches with cobalt, zinc and cadmium efflux proteins (COG3965) [19]. pSymAencoded NP_436297.2 is orthologous to YP_003064775 (E = 3e-30) encoded on the chromosome of 'Ca. Liberibacter asiaticus', but neither has an ortholog encoded by the S. meliloti chromosome (Table 1). SMART analysis of 'Ca. Liberibacter asiaticus' YP_003064775, annotated as a nhaA gene, predicts a sodium/ proton antiporter domain (E = 2.1e-113) at residues 4 to 379 of the 381 amino acid protein [20,21]. These two protein pairs are the only two protein pairs uniquely shared by pSymA and 'Ca. Liberibacter asiaticus'. A third orthologous protein pair comprised of NP_436118.1 (pSymA) and YP_003065337 ('Ca. Liberibacter asiaticus') is much more similar in amino acid sequence (E = 1e-134) than is NP_436118.1 and its ortholog encoded by the S. meliloti chromosome, NP_384912 (E = 1e-22). Bioinformatic analyses revealed that YP_003065337 is a potassium efflux protein.
Control of intracellular pH is ascribed in its domain architecture. SMART analysis of YP_003065337 confidently predicts two domains as follows: an amino-terminal sodium/proton exchanger domain (E = 2.7e-72) from residues 4 to 394 and a nonoverlapping TrkA domain (E = 8.3e-25) for redox regulation, from amino acids 451-566 of the 609 amino acid protein. 'Ca. Liberibacter asiaticus' YP_003065245, a potassium uptake protein, has E = 0 alignments with both pSymA (NP_436237.1) and S. meliloti chromosomal orthologs (Table 1). These proteins likely contribute to the maintainence of intracellular homeostasis through the regulation of pH and the control of mono-and divalent cation concentrations.

Signal Transduction
There are several orthologous proteins encoded by genes shared between pSymA and the 'Ca. Liberibacter asiaticus' and S. meliloti chromosomes that are predicted to function in sensing and signal transduction. For example, YP_003064826, a PAS/PAC sensor signal transduction kinase has a pSymA ortholog and a S. meliloti chromosomal ortholog. Several two-component response regulators or sensor histidine kinases are encoded both by genes on pSymA and the genome 'Ca. Liberibacter asiaticus', and include YP_003065306, which has a characteristic N-terminal CheY-like sensor and Trans_reg_C which are DNA-binding transcriptional activator/effector domains. YP_003064863, a two component sensor histidine kinase/response regulator hybrid protein, is a FixL-like oxygen-controlled histidine kinase homologous to several pSymA-encoded proteins (E = 3e-49 for the most similar ortholog) ( Table 1).

ABC-cassette Type Amino Acid Transporters
ATP Binding Cassette type amino acid transporters comprise the largest class of genes shared by 'Ca. Liberibacter asiaticus', pSymA and the S. meliloti chromosome ( Table 2). For most ABCtype transporter proteins encoded by 'Ca. Liberibacter asiaticus' genes there are multiple homologs on pSymA, dispersed over the megaplasmid (Table S1). YP_003064586 is an example of an ABC-type transporter protein from 'Ca. Liberibacter asiaticus'. In addition to NP_435288.1 there are 10 more pSymA-encoded proteins homologous to YP_003064586 (E = 4e-67 up to 1e- 14), and all are annotated as general amino acid transporters with an ATP binding site and other characteristics of an ABC type amino acid transporter. The corresponding genes are dispersed across the pSymA ( Fig. 1A; Table 2).
YP_003064753 is a proline/glycine betaine ABC transporter protein with eight pSymA-encoded homologs (Table S1). YP_003064754, another proline/glycine betaine permease/ABC transporter permease protein, has two pSymA-encoded homologs. 'Ca. Liberibacter asiaticus' YP_003065117 and YP_003064552, both ATP-binding ABC-type transporter proteins, each have four homologs encoded by genes on pSymA. YP_003065525, a putative amino acid-binding periplasmic protein, is homologous to two pSymA-encoded predicted proteins. YP_003065526, an ABC-type amino acid transporter permease, has 7 homologs encoded by pSymA genes. These are examples of multiple, redundant shared homologs of ABC-type amino acid transport systems encoded by pSymA. 'Ca. Liberibacter asiaticus' YP_003064636 is an exception since this putative transmembrane ABC-type transporter permease has only one pSymA-encoded homolog, NP_435480.1 ( Table 2). The multiplicity of ABC-type transporter homologs on pSymA presumably provides a repertoire of ABC transporters for S. meliloti, and is consistent with a high magnitude of importance for ABC transporter systems in S. meliloti. In contrast, 'Ca. Liberibacter asiaticus' lacks the general overall redundancy of ABC-type transporters present in S. meliloti.
Metabolic cofactors are also likely to be imported by 'Ca. Liberibacter asiaticus' and S. meliloti from the plant host. For example, essential vitamins are up taken by ABC-type transporters encoded by pSymA and the 'Ca. Liberibacter asiaticus' chromo-  some. YP_003064972, an ATP-binding component of an ABC transport complex for thiamine, has two homologs on both pSymA and the S. meliloti chromosome.
Genes on pSymA with orthologs on the 'Ca. Liberibacter asiaticus' and S. meliloti chromosomes also encode enzymes involved in the synthesis of amino acids ( Table 2). The predicted protein products of 'Ca. Liberibacter asiaticus' YP_003064878 and pSymA NP_435501.1 (E = 3e-75) have domains expected of threonine synthase, ThrC, a member of the TrpB tryptophan synthase superfamily. YP_003064845 is an acetylornithine transaminase protein, part of the arginine metabolism and lysine biosynthesis pathways. 'Ca. Liberibacter asiaticus' YP_003064846 and its orthologs in S. meliloti, encoded by genes on pSymA and its chromosome, are predicted to be an ornithine carbamoyl transferase, involved in arginine metabolism. YP_003065195 and its S. meliloti homolog are succinyl diaminopimelate desuccinylase involved in lysine biosynthesis. However, NP_436258.1, a similar protein encoded by pSymA, is annotated as an acetylornithine deacetylase, an enzyme which catalyzes a step in arginine metabolism. Plant hosts of 'Ca. Liberibacter asiaticus' are presumed to supply some amino acids to the obligate phytopathogen since certain genes for the biosynthesis of several amino acids were not found in the comparison of the chromosomes of 'Ca. Liberibacter asiaticus' and S. meliloti [2].

Export of Bioactive Molecules
In addition to the proteins involved in the import or potential export of amino acids and metabolic cofactors, 'Ca. Liberibacter asiaticus' shares genes with pSymA and the S. meliloti chromosomes Table 2. Proteins shared between the Sinorhizobium meliloti plasmid pSymA, and the 'Ca. Liberibacter asiaticus' and Sinorhizobium meliloti chromosomes with roles in ABC-Type transport and the synthesis of amino acids and metabolic cofactors. for several proteins used to export small bioactive molecules (Table 3). These include YP_003064686 an ABC-type transporter with excellent Blast alignments with chromosomal virulence gene (chvD) in Agrobacterium, Bartonella and Brucella [22]. The 'Ca. Liberibacter asiaticus' genome encodes two nodI homologs, likely involved in exporting lipid virulence factors. YP_003065157, an ATP-binding ABC-type transporter protein with a yhbG domain, is similar to pSymA nodI, a multidrug effluxlike protein that secretes lipids and lipopolysaccarides including cell wall and outer membrane or Nod factor components. Interestingly, pSymA encodes six homologs of YP_003065157, consistent with important roles for these proteins in the S. meliloti/ alfalfa interaction. When ABC-type transporters of 'Ca. Liberibacter asiaticus' were blasted against the Transporter Classification Database (http://www.tcdb.org/), YP_003065330, annotated as an ABC-type transporter nucleotide binding/ATPase, was homologous with Q2G2M9 (4.00E-088), a putative multi-drug export ATP-binding/permease protein of Staphylococcus aureus,-SAOUHSC_02003. Like YP_003065157, YP_003065330 aligns best with nodI NP_435718.1 (2.00E-021) encoded by pSymA.
'Ca. Liberibacter asiaticus' YP_003065113, orthologous to pSymA-encoded protein NP_435856.2 (E = 5e-33), a serine protease DO protease, has a PDZ-metalloprotease domain, a PDZ serine protease domain and a trypsin domain, all fused into a single multi-domain protease product. This protein, a probable virulence factor, is likely to protrude from 'Ca. Liberibacter asiaticus' into the host cell where it can presumably digest host proteins using zinc as a cofactor.

Regulation of Transcription and Translation
'Ca. Liberibacter asiaticus', pSymA and S. meliloti share several proteins that regulate transcription (Table 3). YP_003064695 is a member of the LysR family of transcription regulator proteins. Its importance for plant interaction is indicated by at least 14 LysRlike proteins encoded by and distributed across pSymA (E = 1e-21 to E = 1e-10) (Fig. 1C; Table S1). Other genes encoding transcriptional regulators are not as extensively duplicated on pSymA, consistent with LysR family transcription regulators being of unique importance in plant interaction in S. meliloti [23]. The 'Ca. Liberibacter asiaticus' genome, by contrast, encodes only YP_003064695, annotated as transcriptional regulator with a LysR region. YP_003065329, a putative sigma-54-dependent transcription regulator protein is similar to NP_435689 in pSymA annotated as NifA. NifA is a DNA-binding ATPase and CheY-like two-component sensor-based response regulator that is known to  -18), and is involved in the synthesis of proteins. YP_003065391 encodes aspartyl/glutamyl-tRNA amidotransferase, an enzyme known to allow formation of correctly charged asparagine or glutamine tRNAs in organisms which lack either or both a asparaginyl-tRNA synthetases or a glutaminyl-tRNA synthetase.

Electron Transfer
An important conserved microsyntenous orthologous gene (MOG) cluster, occurs in the 'Ca. Liberibacter asiaticus' genome and in the genomes of four other members of the Rhizobiales that were analyzed previously [26]. This conserved MOG encodes thirteen subunits that comprise a ''mitochondrial-like'' Type I respiratory complex (Table 5). Genes encoding this respiratory complex are also found on pSymA, and orthologous proteins specified by 'Ca. Liberibacter asiaticus' chromosomal genes include the following: Chain A, NuoA2 NADH: ubiquinone oxidoreductase YP_003065265 and NP_436080.1 (E = 9e-14); Chain B, or NuoB2 YP_003065266 and NP_436079 (E = 6e-48); Chain C, or NuoC2 YP_003065267 and NP_436078.1 (E = 6e-33); Chain D, or NuoD2 YP_003065268 and NP_436077.1 (E = 1e-110); Chain E or NuoE2, YP_003065269 and NP_436076.1 (E = 4e-24) and Chain F or NuoF2 YP_003065271 and NP_436075.1 (E = 1e-104). Chains G, H and I/J or NuoG2, NuoH2, a multi-subunit ubiquinone oxidase have the following orthologs: YP_003065272 and NP_436074.1 (E = 2e-62); YP_003065273 and NP_436071.1 (E = 1e-51), YP_003065274 and NP_436072.1 (E = 1e-22), and YP_003065275 and NP_436087.2 (E = 8e-11). YP_003065278 is orthologous with pSym proteins NP_436085.1 and NP_436083.4 annotated as, respectively, a multidomain K/L oxidoreductase, a NADH ubiquinone oxidase (COG1009), or a multi-domain Na+/ K+ antiporter, largely synonomous, involved in energy generation, cation efflux or intracellular pH control. 'Ca. Liberibacter asiaticus' YP_003065279 and pSymA NP_436082.1 (E = 2e-92) are chain M of ubiquinone oxidoreductase. 'Ca. Liberibacter asiaticus' YP_003065280 and pSymA NP_436081.1 are chain N (E = 4e-57). Nearly all orthologous proteins shared between pSymA and S. meliloti chromsome have lower E-values aligned with each other than do orthologs shared between pSymA and the 'Ca. Liberibacter asiaticus' chromosome (Tables 1-4). However the amino acid sequence similarities between protein components of the respiratory complex I encoded by pSymA, and the 'Ca. Liberibacter asiaticus' genome and the S. meliloti chromosome are highly comparable with each other ( Table 5). The organization of the genes in genomic regions encoding the NADH dehydrogenase complex is the same for S. meliloti and 'Ca. Liberibacter asiaticus' (Fig. 2). NuoG and NuoL are encoded by genes in the expected cluster on the S. meliloti chromosome. But the e-values obtained by pBlast of pSymA NuoG and NuoL vs S. meliloti are better for genes found elsewhere on the S. meliloti chromosome. The best match for pSymA NuoG (NP_43074) on the S. meliloti chromosome is a NAD-dependent formate dehydrogenase alpha subunit (NP_387115; E = 2e 295 ). The NADH dehydrogenase subunit G (NP_385378; E = 8e 277 ) is the second best result. This protein is transcribed and translated from a gene with the expected position in the NADH dehydrogenase MOG and aligns well with the 'Ca. Liberibacter asiaticus' NuoG (e = 0). A similar finding was obtained when pSymA NP_436085 encoding the NuoL subunit was used in a blast search of the chromosome of S. meliloti. The best matches were to proteins annotated as monovalent cation/H+ antiporter subunits located at different positions on the chromosome. NADH dehydrogenase subunit L (NP_385383; E = 7e 225 ) encoded by the chromosome was significantly less well-aligned. As was seen for NuoG, this protein is transcribed from a gene with the expected position in the NADH dehydrogenase MOG and aligns well with the 'Ca. Liberibacter asiaticus' NuoL (e = 0). Thus these proteins likely function in the role of NuoL. The NADH dehydrogenase gene cluster on pSymA has a different arrangement of genes (Fig. 2). Genes encoding NuoA-NuoG in the center of the Nuo gene cluster are flanked on one side by genes encoding NuoJ -NuoN and by genes, transcribed from the opposite strand, that encode NuoH and NuoI on the other side.
Other proteins are encoded by genes shared by 'Ca. Liberibacter asiaticus', pSymA and the chromosome of S. meliloti, and have roles in energy generation. YP_003065437, is a probable electron transfer flavoprotein-quinone oxidoreductase or FixC. YP_003064703 is orthologous with both pSymA-borne NP_435265.2 and NP_435950.1, which are zinc-dependent NADPH quinone oxidoreductases, COG0604, and are important in energy production. YP_003064781 and YP_003064782, encoded by adjacent genes, are orthologs of NP_436008.1 and NP_436007.1 (E = 3e-73 and 2e-77 respectively). Both genes have two adjacent ortholog pairs on both pSymA and the S. meliloti chromosome. YP_003065174 and NP_435375.1 (E = 3e-76) are annotated as energy yielding FAD-dependent dehydrogenases. YP_003065041 is coproporphyrinogen III oxidase and is orthologous to a pSymA-borne locus NP_435937.1. The protein has two domains; N-terminal Radical-SAM super family and a Cterminal HemN_C superfamily, which obtains electrons from a flavodoxin-like system regenerated by a nicotinamide cofactordependent flavodoxin [27]. 'Ca. Liberibacter asiaticus' YP_003064696 is a NADPH thioredoxin reductase orthologous to NP_436285.1.

Discussion
The premise for identifying protein products of 'Ca. Liberibacter asiaticus' orthologous to those encoded by genes on pSymA was an hypothesis that such genes and their protein products in all likelihood play direct or indirect role(s) in establishing and maintaining an intimate intracellular interaction with eukaryotes. 'Ca. Liberibacter asiaticus' has an obligatory requirement for a   eukaryotic host, and there are multiple orthologs of these genes in Sinorhizobium meliloti. These factors preclude mutagenesis studies at the present time to test this hypothesis. In particular, we discovered that there are several orthologous genes in the 'Ca. Liberibacter asiaticus' genome and on pSymA that encode proteins with vital roles in maintaining intracellular homeostasis for monoand divalent cations and pH. Genes encoding a Na + /H+ antiporter [20,21] and a cation efflux antiporter protein that secretes excess metal ions Cd ++ , Zn ++ or Co ++ in exchange for K + and H + [19,28] were identified as orthologs shared exclusively by pSymA and 'Ca. Liberibacter asiaticus,' i.e., not found on the S. meliloti chromosome. In a previous study we compared the proteins encoded by the circular chromosomes of 'Ca. Liberibacter asiaticus' and four other members of the Rhizobiales. In that study the cation efflux antiporter (YP_003064907) was found to be uniquely shared between 'Ca. Liberibacter asiaticus' and phytopathogenic Agrobacterium tumefaciens C58 (NP_354015; 3E-84). The Na + /H + antiporter (YP_003064775) shared between pSymA and 'Ca. Liberibacter asiaticus' is also shared with 10 species or strains of the Bartonellaceae (6E-60-4E-48), but not with the other members of the Rhizobiales studied [26]. This suggests that this protein is used to facilitate an intracellular lifestyle. In addition, another protein encoded by genes shared by pSymA and the 'Ca. Liberibacter asiaticus' and S. meliloti chromosomes, is a glutathi-one-gated potassium efflux protein that exchanges potassium for sodium and protons and thereby modulates cytoplasmic pH [29,30]. This orthologous protein pair encoded by both pSymA and the 'Ca. Liberibacter asiaticus' genome align with an e-value that is 110 orders of magnitude lower than the alignment of the orthologous proteins encoded by pSymA and the S. meliloti chromosome. Thus, the maintenance of intracellular pH and ion homeostasis are likely critical functions of proteins encoded by genes shared uniquely by 'Ca. Liberibacter asiaticus' and pSymA (but not the S. meliloti chromosome). Other 'Ca. Liberibacter asiaticus' genes with orthologs on pSymA likely encode proteins with either direct or indirect nutritional or energy roles in establishing and maintaining an intimate intracellular interaction with eukaryotic hosts. We identified 86 'Ca. Liberibacter asiaticus' genes with 182 probable orthologs on pSymA and the S. meliloti chromosome. Most of the orthologous proteins encoded by pSymA and the S. meliloti chromosome produced alignments with substantially more significant E-values than orthologous proteins encoded by 'Ca. Liberibacter asiaticus' and pSymA. Individual genes encoded by the reduced genome of 'Ca. Liberibacter asiaticus' may have many orthologs or paralogs carried by the pSymA megaplasmid besides additional similar genes on the chromosome of S. meliloti. An example is YP_003064948, a 3-ketoacyl-(acyl-carrier-protein) reductase with 20 similar proteins encoded by pSymA. Thus, numerous physiological and niche adaptations conferred upon S. meliloti by pSymA may not be available to 'Ca. Liberibacter asiaticus.' However, in a reduced genome such as that of 'Ca. Liberibacter asiaticus', genes that are retained may function with relaxed substrate specificity [31,32], compensating for the lack of multiple homologs in the 'Ca. Liberibacter asiaticus' genome. The multiplicity of pSymA orthologs complicates any mutagenesis strategy to definitively establish their roles in S. meliloti. Protein products not constitutively expressed may be considered as candidates for being involved with niche specialization or pathogenesis; S. meliloti has conditionally expressed genes that are especially important for nitrogen fixation and nodulation symbiosis. YP_003064695 is a putative regulator of transcription by 'Ca. Liberibacter asiaticus', with 14 related proteins in pSymA, many of which are annotated as LysR transcriptional regulatory proteins. Despite considerable conservation both structurally and functionally, LysR-type transcriptional regulators regulate a diverse set of genes, including those involved in virulence, metabolism, quorum sensing and motility [33,34]. In S. meliloti, LysR proteins function in exquisite control of a sequence of events leading to root colonization, initiation of nodulation and subsequent differentiation of nodules containing S. meliloti [23]. Fatty acid products of the 3-ketoacyl-(acyl-carrier-protein) reductase orthologs are also used to complete the structure of nodulation factors. This may be why the extensive collection of LysR regulatory elements and 3-ketoacyl-(acyl-carrier-protein) reductases encoded on pSymA are not found on the 'Ca. Liberibacter asiaticus' chromosome. Thus, 'Ca. Liberibacter asiaticus' may have much less capability for finely nuanced control of the expression of genes, consistent with its obligatory intracellular lifestyle. Parasitism of citrus by 'Ca. Liberibacter asiaticus' follows direct injection of the pathogen into the host and results in systemic invasion of phloem tissues throughout the host [9,10], and differentiated nodules are not produced.
Genes with orthologs on both pSymA and the chromosome of S. meliloti are largely specialized for symbiosis and nitrogen metabolism in S. meliloti and include 48 ABC-type transporter genes. The genome of 'Ca. Liberibacter asiaticus' encodes only 11 ABC-type transporter proteins that are orthologous to pSymA-encoded proteins, compared with a total of about 40 ABC-type transporters encoded by the chromosome [2]. These transporters likely serve to import sugars, amino acids and precursors and purines and pyrimidines into 'Ca. Liberibacter asiaticus' from the host. The genome of 'Ca. Liberibacter asiaticus' is known to be deficient in genes used in the biosynthetic pathways of these compounds [2,12].
ABC-type transporters may also be used to export small molecules by 'Ca. Liberibacter asiaticus'. YP_003064989 is an ABC-type transporter protein encoded by 'Ca. Liberibacter asiaticus' that likely functions to export glucans for osmoprotection. A class of proteins that confer multiple drug resistance, are called 'RND' for Resistance, Nodulation and Division [35]. RNDtype proteins confer drug resistance based on rapid efflux of drugs from the bacterial cell. YP_003065157, is an ATP-binding ABCtype transporter protein with a yhbG domain, and is orthologous to pSymA NodI, a protein that is required for nodulation of alfalfa by S. meliloti. NodI-like proteins secrete lipids and lipopolysaccarides such as cell wall, outer membrane or Nod factor components needed to stimulate the differentiation of nodules by the host. Very interestingly pSymA encodes six homologs of YP_003065157. Orthologs of ABC-type transporters encoded by 'Ca. Liberibacter asiaticus' were identified with the ''Transporter Classification Database'' (http://www.tcdb.org/). YP_003065330, annotated as an ABC-type transporter with nucleotide binding and ATPase domains was homologous to Q2G2M9 (4.00E-88), annotated as a putative multi-drug export ATP-binding/permease protein, Staphylococcus aureus SAOUHSC_02003. YP_003065330 aligns best with nodI NP_435718.1 (2.00E-021) encoded by pSymA. Thus these proteins at the least share similar structural motifs.
Genes encoding type IV pili may have evolved from structures used for DNA transfer but it is now known that virulence proteins may be secreted by components of a type IV pili [36]. Genes encoding type IV pili are shared between 'Ca. Liberibacter asiaticus', S. meliloti, and pSymA. Effectors directly involved with the establishment of 'Ca. Liberibacter asiaticus' as an intracellular pathogen living within the phloem cells of citrus, may be transferred from the bacterium to its host by type IV secretion involving pilus assembly proteins. If pili are produced by 'Ca. Liberibacter asiaticus', they may also have roles in twitching motility and biofilm formation as in Xylella fastidiosa [37].
Why is the entire S. meliloti chromosomal respiratory complex I gene set duplicated on pSymA? Plants respond to bacterial pathogens by producing a sustained oxidative burst leading to apoptosis. The respiratory complex protein NuoG can also be used by Mycobacterium tuberculosis to pump protons out of the cell to neutralize such superoxide radicals [38]. This may explain the duplication of the genes encoding the respiratory complex on pSymA and provide an additional role for NuoG in the 'Ca. Liberibacter asiaticus'/citrus interaction. Homeostasis with respect to sodium is also of critical importance to both S. meliloti and 'Ca. Liberibacter asiaticus' as discussed above, and in addition to pumping protons out of the cell to create the proton motive force needed for the synthesis of ATP, respiratory complex I of at least some Gram negative bacteria can also transport sodium ions out of the cells [39]. Besides an obvious potential gene dosage effect, redundant copies carried on an extrachromosomal element provide a palette for gene evolution. However in spite of the potential for diversification of these genes afforded to pSymA, substantial diversification is not apparent since the E-values for the orthologs encoded by pSymA, 'Ca. Liberibacter asiaticus' and S. meliloti are very similar.
Most 'Ca. Liberibacter asiaticus' protein products that align significantly with proteins encoded by pSymA also align significantly with predicted protein products encoded by the S. meliloti chromosome. Thus, although present as single copies for the most part in 'Ca. Liberibacter asiaticus', these genes are present in multiple copies per replicon in the free-living microsymbiont. The presence of multiple orthologs in pSymA of 'Ca. Liberibacter asiaticus' proteins such as the amino acid transporter YP_003064586, the fatty acid dehydrogenase YP_003064948 and the LysR transcriptional regulator YP_003064695, suggests that their products are of particular importance in host/microbe interactions, and are likely to be examples of a single protein acquiring multiple functions in an organism with a reduced genome [32]. 'Ca. Liberibacter asiaticus' very notably also has two or three genes shared with pSymA but not the S. meliloti chromosome. These genes are predicted to produce proteins that generate a proton gradient with ATP-producing potential, maintain sodium and potassium homeostasis, prevent over accumulation of divalent metal cations and maintain intracellular pH. Thus the maintenance of intracellular homeostasis is potentially a vital requirement for successful colonization of citrus and psyllids by 'Ca. Liberibacter asiaticus'. In an obligate pathogen, cellular vitality and pathogenesis are tied together.

Materials and Methods
The genome of 'Ca. Liberibacter asiaticus' was compared with both the circular chromosome and megaplasmids, especially pSymA, of S. meliloti. To identify orthologous proteins, predicted amino acid sequences ('Ca. Liberibacter asiaticus' strain psy62, RefSeq NC_012985; Sinorhizobium meliloti strain 1021 RefSeq NC_003047, and pSymA RefSeq NC_003037) were downloaded from NCBI. Using default BLAST parameters, each predicted amino acid sequence from the ORFs identified on pSymA of the S. meliloti 1021 genome was BLASTed against the predicted amino acid sequences of the ORFs on the chromosomes of 'Ca. Liberibacter asiaticus' and Sinorhizobium meliloti strain 1021. Perl scripts and Excel spreadsheets were created to identify hits between genomes with low, negative e-values and to extract annotations from Genbank. In addition, amino acid similarity and length of the blast hit was extracted from each top hit from the BLAST output. First, a general analysis was done extracting predicted protein products having BLAST alignment values of e -10 or lower. Similar or homologous proteins were also identified manually where possible to be consistent with the annotations from the authors of each respective genome. That is, annotations of ORFs were extracted from NCBI annotations of the genome of 'Ca. Liberibacter asiaticus' and megaplasmid pSymA of Sinorhizobium meliloti strain 1021, respectively. Thus, the matching homolog might not have the best e-value or match with the top hit as many proteins share the same domain structure or amino acid similarity, but are functionally quite different. Similarly, for proteins of interest that were unique to each genome, proteins were sorted for positive e-values, signifying that an orthologous protein was not encoded in a respective genome. Orthologous genes from the 'Ca. Liberibacter asiaticus chromosome, the S. meliloti chromosome and megaplasmid pSymA were mapped on linear representations of the respective genophores opened at their origins of replication.

Supporting Information
Table S1 Proteins shared between the Sinorhizobium meliloti plasmid pSymA, and the 'Ca. Liberibacter asiaticus' and Sinorhizobium meliloti chromosomes. The protein IDs and annotations used by NCBI and the e-values of pairwise comparisons are provided. (XLSX)