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Microbiology and Molecular Biology Reviews, June 2002, p. 272-299, Vol. 66, No. 2
1092-2172/02/$04.00+0     DOI: 10.1128/MMBR.66.2.272-299.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Whole-Genome Analysis of Transporters in the Plant Pathogen Xylella fastidiosa

Joao Meidanis,1 Marilia D. V. Braga,1 and Sergio Verjovski-Almeida2*

Instituto de Computação, Universidade de Campinas, Campinas, São Paulo 13083-970,1 Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo 05508-900, Brazil2

SUMMARY
INTRODUCTION
SEQUENCE ANALYSIS
QUANTITATIVE COMPARISON WITH OTHER MICROORGANISMS
    Major Transporters
    Additional Transporters
TRANSPORTERS PRESENT IN X. FASTIDIOSA
    Alpha-Type Channels
    ß-Barrel Porins
    Pore-Forming Toxins
    Electrochemical Potential-Driven Transporters
        Symporters, antiporters, and uniporters.
        (i) Major facilitator superfamily.
        (ii) Other secondary porters.
    Energizers
    Primary Active Transporters
        Transport driven by phosphate bond hydrolysis.
            (i) ABC superfamily.
            (ii) Other phosphate bond-driven transporters.
        Oxidoreduction-driven transporters.
    Phosphotransferase System
    Auxiliary Proteins
    Unknown and Uncharacterized Transporters
CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES

   SUMMARY
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The transport systems of the first completely sequenced genome of a plant parasite, Xylella fastidiosa, were analyzed. In all, 209 proteins were classified here as constitutive members of transport families; thus, we have identified 69 new transporters in addition to the 140 previously annotated. The analysis lead to several hints on potential ways of controlling the disease it causes on citrus trees. An ADP:ATP translocator, previously found in intracellular parasites only, was found in X. fastidiosa. A P-type ATPase is missing—among the 24 completely sequenced eubacteria to date, only three (including X. fastidiosa) do not have a P-type ATPase, and they are all parasites transmitted by insect vectors. An incomplete phosphotransferase system (PTS) was found, without the permease subunits—we conjecture either that they are among the hypothetical proteins or that the PTS plays a solely metabolic regulatory role. We propose that the Ttg2 ABC system might be an import system eventually involved in glutamate import rather than a toluene exporter, as previously annotated. X. fastidiosa exhibits fewer proteins with >=4 {alpha}-helical transmembrane spanners than any other completely sequenced prokaryote to date. X. fastidiosa has only 2.7% of all open reading frames identifiable as major transporters, which puts it as the eubacterium having the lowest percentage of open reading frames involved in transport, closer to two archaea, Methanococcus jannaschii (2.4%) and Methanobacterium thermoautotrophicum (2.4%).


   INTRODUCTION
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Xylella fastidiosa is a plant-pathogenic bacterium that causes variegated chlorosis in citrus trees and other diseases in a wide range of plant hosts (15, 34). The citrus pathogen has an insect as its vector, which takes the parasite from tree to tree while feeding on the xylem. Among the plant-pathogenic bacteria, X. fastidiosa is the first whose genome has been completely sequenced (44). Annotation of this genome was done by several groups, each focusing on a particular functional category. The categories were based on the classification proposed by Riley (38) for Escherichia coli. In this effort, we were responsible for the transport category, and it soon became clear that in the scope of the whole genome paper (44), there was no room to describe all the interesting facts we were finding. For this reason we decided to produce a detailed separate inventory of all transport proteins and the conclusions associated with these findings.

Apart from being the first plant-pathogenic bacterium to be sequenced, X. fastidiosa was also one of the least known organisms targeted for sequencing. At the beginning of the sequencing project (1998), only six Xylella sequences were deposited in GenBank (http://www.ncbi.nlm.nih.gov/). The sequencing project made X. fastidiosa jump from almost unknown to almost completely known. However, very few, if any, of the functional predictions made for Xylella proteins were verified in the laboratory. This is, as far as we know, the status of all the proteins described here: all predictions were made based solely on computational evidence. In this paper we made an effort to suggest experiments that would contribute to verifying some of the most important claims made here.

In addition, many of the important processes of X. fastidiosa related to the diseases it causes probably rely on proteins—hemolysins, adhesins, xanthum gum-producing enzymes, virulence factors, detoxification enzymes—involved in one way or another with transport, which provides additional interest for the study of the important class of transport proteins.


   SEQUENCE ANALYSIS
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The list of all X. fastidiosa predicted proteins was obtained from the sequencing project web site (http://www.lbi.ic.unicamp.br/xf). The number of transmembrane segments (TMSs) of each Xylella protein was estimated using PSORT (25). For comparison, PSORT (25) was used for the prediction of the number of TMS for each of the 18 completely sequenced bacteria that had been analyzed previously by Paulsen et al. (31) as well as for Saccharomyces cerevisiae, which has been analyzed by Paulsen et al. (30). For identification of transport families, each Xylella sequence was compared to a database consisting of all examples appearing in M. Saier's transport protein Classification web site (http://www-biology.ucsd.edu/{approx}msaier/transport). This site contains, for each category of transport proteins, a detailed description, including the mode of transport, protein topology, substrate specificity, and species specificity, followed by extensive literature and, more important for our purposes, examples of transport proteins with SWISS-PROT and GenBank reference codes. The site is constantly being updated, so we had to freeze a local copy for our work. We downloaded all relevant pages, corrected some ill-formed reference numbers, fetched all example sequences from the public databases, manually updated the few changes that occurred, and froze our local transport protein database on 9 April 2001.

The comparison consisted of a first phase, in which we used Blast2 (2) and queried all Xylella sequences against the transport protein database using default parameters, except that the Blastp search was done without a low-complexity filter, followed by a second phase, in which PRSS (32) was used to evaluate the significance of the sequence similarity score and to check for the absence of sequence composition bias. It is known that {alpha}-helical transmembrane domains are rich in L, V, I, and F amino acids and that there is a high repetition rate of these four amino acids in the 18- to 20-amino-acid transmembrane stretches. As a result, a considerable number of putative transmembrane domains are usually filtered out of a Blastp search done with a low-complexity filter. Filtering the transmembrane domains may cause the search to miss important transport family similarities. Thus, search of Xylella proteins against the transport protein examples was done using Blastp without a low-complexity filter, and matches having an e value lower than 0.01 were selected in the first phase of the search.

In a second phase we used the homology assessment program PRSS from the FASTA package (32) to confirm that the hits pointed out by Blast2 without a filter were not caused by sequence composition bias. In PRSS we used 200 random shuffles with a local window of 10 amino acid residues and admitted as "good matches" hits that got a score of 0.00001 or lower. Blast E values and PRSS scores are shown in Tables 2 to 7 and are shown in the extended format of Table 2, which is available at http://onsona.lbi.dcc.unicamp.br/{approx}meidanis/. By selecting the 283 Xylella proteins that had a PRSS score of 0.00001 or lower and applying a few manual-scrutiny criteria described below, the list was reduced to 209 transport proteins. It can be seen in Tables 2 to 7 that the highest Blast E value (weakest hit) is 3e-04, which was found for two proteins, XF0437 (1.A.23.1.2) and XF1400 (9.A.19.1.1). It should be noted that nine proteins (XF0103, XF0268, XF0563, XF0777, XF1404, XF1496, XF1978, XF2287, and XF2301) which passed the analysis described above and are included in Tables 2 to 7 do have E values higher than 3e-04 when Blast search against the transport protein database is run with a low-complexity filter. We have placed a remark for each of them in the Comments column in Tables 2 to 7, showing the Blast E value with a filter.


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  		TABLE 2. Channels, ß-porins, and pore-forming toxinsa

 

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  		TABLE 7. Xylella transporters of unknown classification, uncharacterized transporters, and hypothetical and undefined transportersa

 
With the good matches, we constructed a preliminary list of transport-related Xylella proteins, and each Xylella protein automatically acquired one or more TC numbers, inherited from the hits in the examples database. The preliminary list was scrutinized by us, and as a result the final number of transporters was reduced from 283 to 209, as described above. The criteria used included the following. (i) Some proteins were eliminated mainly because they had a much better hit with some protein of another, nontransport function, or because the alignment with the transport example did not cover at least 50% of the sequence described in the transport classification site. In some instances, matches with less than 50% coverage of the example were not eliminated because either a specific conserved domain that is described in the literature as a signature of the corresponding example was also present in the Xylella protein or the putative gene was clustered with neighboring Xylella genes of the same family that had good matches with TC examples. (ii) When a Xylella protein was similar to examples from more than one TC category, a decision had to be made as to which one was most probable. This decision took into account E values from Blast hits and PRSS runs, coincident number of predicted TMSs, prior annotation available from the Xylella genome project, proximity to other related proteins in the genome (in the case of ABC transporters, for instance), and scientific literature, including the very well documented transport classification site.


   QUANTITATIVE COMPARISON WITH OTHER MICROORGANISMS
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Table 1 contains a summary of the number of X. fastidiosa proteins predicted by the PSORT algorithm (25) to have a specific number of TMSs; they were separated among six classes with different TMS ranges. For comparison, we used PSORT (25) to predict the number of TMSs for another 18 completely sequenced bacterial genomes that have been analyzed previously by Paulsen et al. (31) and for S. cerevisiae, which was analyzed by Paulsen et al. (30). It can be seen that X. fastidiosa has 957 proteins with at least one putative TMS. A considerable number of transmembrane proteins will have the potential to be transporters. Among them, 170 proteins (6% of all Xylella proteins) have four or more TMSs (Table 1). Typically, in the organisms examined to date, 40 to 60% of the proteins with four or more TMSs are associated with transport (31).


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  		TABLE 1. Transmembrane proteins in X. fastidiosa, 18 other prokaryotes, and S. cerevisiaea

 
It is interesting that X. fastidiosa is the prokaryote with the smallest percentage (6%) of proteins with four or more TMSs (Table 1), which is in agreement with the fact that only 75 major transporters (2.7% of all proteins) were identified in X. fastidiosa (see below). Therefore, X. fastidiosa is the eubacterium with the smallest proportion of major transporters (2.7%) among the fully sequenced bacteria that were analyzed (Table 1). Only two archaea, Methanococcus jannaschii and Methanobacterium thermoautotrophicum, have a lower percentage of transporters (2.4%).

Major Transporters

In order to permit a direct comparison of the number of Xylella transporters with the 18 previously analyzed fully sequenced bacteria (31), we have opted to use the same classification strategy used by Paulsen et al. (31). In that work, the transport families that were used for computing the number of transport systems only included {alpha}-helical channel proteins and did not include the ß-porins and pore-forming toxins (31). All electrochemical potential-driven transporters (secondary transporters) were included, but only some of the primary active transporters driven by phosphate bond hydrolysis were included, namely the ABC transporters and F- and P-type ATPases; no type II, III, or IV transport systems or oxidoreduction-driven active transporters or phosphotransferase (PTS) systems were counted (see Table 3 in reference 31). In the same manner, the so-called unclassified transporters were counted (TC 9.A); however, the energizers (TC 2.C), auxiliary transporters (TC 8) (39), and putative uncharacterized transporters (TC 9.B) were not counted (31). We have opted here to call major transporters all those transporters computed in Paulsen et al. (31), and for X. fastidiosa to compute separately both the major transporters (Table 1) and all the transport families (Tables 2 to 7 [shown below]) that could be identified by comparison to transport family examples available in Saier's phylogeny-based transport protein classification web site (http://www-biology.ucsd.edu/{approx}msaier/transport).

Based on the major transport protein families analyzed by Paulsen et al. (31), we have identified 106 Xylella proteins which comprise 74 major transporters belonging to 57 families as follows: 5 channels (5 proteins), 39 secondary transporters (44 proteins), 24 primary transporters (51 proteins), and 6 unclassified transporters (6 proteins). The number of major transporters in X. fastidiosa is similar to those found for Archaeoglobus fulgidus (71 transporters), an archaea, and Thermotoga maritima (86 transporters), a deep-branching bacterium (Table 1). The number of major transporters per megabase of DNA is 28 in X. fastidiosa. As pointed out by Paulsen et al. (31), this is a fairly constant value for the other 18 prokaryotes analyzed (average value of 36 transporters per Mb); the conspicuous exceptions are Escherichia coli, Bacillus subtilis, and Haemophilus influenzae, which have 66, 63, and 52 transporters per Mb, respectively.

The 39 secondary transporters of X. fastidiosa mentioned above correspond to 53% of the major transporter types found in this bacterium. Primary transporters represent 32%, while channels and unclassified transporters account for the remaining 15%. Predominance of secondary transporters over primary ones is in accordance with the fact that X. fastidiosa is an aerobic prokaryote in which the respiratory chain is complete. Respiration probably provides most of the energy for generation of a proton motive force that is subsequently used by secondary transporters to effect solute translocation across the membranes. This correlates with previous observations showing that prokaryotic organisms that depend primarily on substrate-level phosphorylation show the highest percentage of primary transporters, while the more aerobic prokaryotes show the reverse tendency (31).

Additional Transporters

In addition to the major transporters described above, we were able to further identify 103 Xylella proteins with similarity to other transporters present in Saier's classification (39). They comprise 64 transporters belonging to 34 families: 25 porins and pore-forming toxins (25 proteins), 10 energizers (10 proteins), 8 active transporters (47 proteins), 10 auxiliary transporters (10 proteins), and 11 putative uncharacterized transporter proteins. Thus, a total of 92 different transport families comprising 139 transporters were identified. In all, 209 proteins were classified as constitutive members of transporter families.

It should be noted that there were an additional 45 Xylella proteins with four or more putative TMSs which are classified as hypothetical or conserved hypothetical (marked H in Table 7) because they do not match the transport protein database and in addition they either do not match any other GenBank protein or match proteins annotated as hypothetical. Another 10 Xylella proteins are classified as undefined because they have four or more TMSs and again do not match the transport protein database; however, they are similar to proteins of other organisms that are given a defined name (see Annotation column for proteins in TC family marked U in Table 7) but have undefined or poorly characterized function (marked U in Table 7). Some 14 of those 55 proteins mentioned above have between 8 and 11 TMSs, and these include many putative transporters, such as an uncharacterized predicted permease (XF0250) with 10 TMSs and unknown function, which has 139 other similar members, all of unknown function, that appear in 16 different bacteria. There is a good chance that of the 55 Xylella proteins with unknown function and four or more TMSs, some 22 to 33 additional hypothetical or undefined proteins (40 to 60%) are Xylella transporters. It should be interesting to concentrate on experiments that would characterize some of those possible transporters.


   TRANSPORTERS PRESENT IN X. FASTIDIOSA
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Tables 2 through 7 summarize our findings about all transport proteins identified in X. fastidiosa. The tables are divided according to the transport family categories described by Saier (39) and provide information about the transport protein example that matches each Xylella protein, such as the Transport Category (TC), the putative substrate transported, the name of the example gene, and the E score and PRSS value of the match. In addition, the original Xylella annotation (44) is shown along with the cluster of orthologous groups (COG) annotation (46) from http://www.ncbi.nlm.nih.gov/COG/. An additional Table 2 (extended) is available at http://onsona.lbi.dcc.unicamp.br/{approx}meidanis/; it has more extensive information that we collected about each match between the Xylella protein and the transport example protein.

Alpha-Type Channels

X. fastidiosa possesses only four channel systems (Table 2). One of them is an interesting member of the voltage-gated ion channel superfamily (1.A.1). The Xylella protein is similar to other putative voltage-gated channels widely found in eukaryotes and so far only found in Deinococcus radiodurans and Pseudomonas aeruginosa bacteria. It has significant similarity to the Shal2 voltage-gated potassium channel from Drosophila melanogaster (1.A.1.2.3, P17971), a member of the voltage-regulated ion transport family (pfam 00520) (http://pfam.wustl.edu/). In the Drosophila protein, transmembrane segment S4 is probably the voltage sensor and is characterized by a series of positively charged amino acids at every third position (48). An identical motif is found in the corresponding transmembrane segment of the Xylella protein, as well as in the channel proteins of the other two bacteria. It will be interesting to characterize the functional role in X. fastidiosa of such a voltage-regulated ion channel.

Protein XF2267 belongs to the major intrinsic protein family (1.A.8) and is a glycerol facilitator (1.A.8.1.1). There is a protein that serves as a large conductance mechanosensitive channel (1.A.22; MscL), which transports ions nonspecifically, with some selectivity for cations over anions. Large-conductance mechanosensitive channels have been shown to release proteins such as thioredoxin and to protect bacteria from cell lysis during an osmotic downshift (27). In addition, two proteins make up small-conductance mechanosensitive ion channels (1.A.23; MscS) which open in response to pressure changes during osmotic downshift just below those that cause cell disruption and death (22).

It is interesting that no one member of the 15 distinct families of holins are present in X. fastidiosa. Holins are channel proteins that organize as homooligomeric complexes that form transmembrane pores which provide the passive transport of murein hydrolases across the cytoplasmic membrane to the cell wall, where these enzymes hydrolyze the cell wall polymer as a step leading to cell lysis (50). In X. fastidiosa, no murein hydrolases were found, and phage-related cell wall degradation and lysis are mediated through phage-related lysozymes (XF1564 and XF1669), which apparently have other mechanisms of secretion that are independent of holins.

ß-Barrel Porins

X. fastidiosa possesses 21 outer membrane proteins (Table 2) that act in conjunction with cytoplasmic membrane transporters to effect the transport of substances from the cytoplasm to the cell exterior.

One of them is an outer membrane factor (1.B.17), which can operate in conjunction with both ATP-binding cassette (ABC) and resistance-nodulation-cell division (RND) transport systems linked to the outer membrane factor protein by a membrane fusion protein (8.A.1). The several proteins involved in this apparatus result in a structure that spans the cytoplasmic membrane, the periplasmic space, and the outer membrane and transports substances directly from the interior of the cell to the outside in one energy-coupled step. This system is used in X. fastidiosa, as in most other bacteria in which it is found, for export of multiple drugs, heavy metals, and bacteriocins (28). X. fastidiosa has only one outer membrane factor protein, but three membrane fusion proteins that function with RND porters (TC 8.A.1.2), two membrane fusion proteins that function with ABC porters (TC 8.A.1.3), and several RND and ABC systems that export drugs.

X. fastidiosa possesses an outer membrane auxiliary protein (1.B.18), annotated as GumB, which works with two other proteins for the export of exopolysaccharides. One of these extra proteins (GumC) spans the cytoplasmic membrane twice and has an ATP-binding domain, as in several other gram-negative bacteria (29). This is a member of the family of cytoplasmic membrane-periplasmic auxiliary-1 proteins with a cytoplasmic (C) domain (TC 8.A.3) and is located in the genome adjacent to the gene encoding the outer membrane auxiliary protein. The third protein (GumJ) is a member of the polysaccharide transporter family (9.A.1), the gene for which is also close to the other two in the genome. In general, polysaccharide transporter family proteins have 12 TMSs, but the Xylella homolog has only nine. A thorough description of the gum-producing machinery of X. fastidiosa is under preparation (F. R. Da Silva, personal communication).

Two members of the secretin family (TC 1.B.22) export proteins and fimbrial structures. A member of the fimbrial usher porin family (TC 1.B.11) also exports fimbriae. Two proteins of the Pseudomonas outer membrane porin family (TC 1.B.5) export phosphate or pyrophosphate.

Nine outer membrane receptors (1.B.14), which use energy produced by the TonB systems (TC 2.C.1), import a variety of substances into the periplasm. These substances find their way into the cytoplasm mainly via ABC transporters. Based on similarities with homologs from other species, we predict that iron siderophores and vitamin B12 are among the substrates imported by this mechanism. Two of the open reading frames (ORFs) classified as vitamin B12 receptors (XF0550 and XF2237) have shown much higher similarity to Schwanella predicted proteins of unknown function.

There is one protein of the OmpA-OprF porin family (TC 1.B.6.1.2), a large family that includes the functionally well characterized OmpA porin of E. coli as well as the OprF porin protein F of Pseudomonas aeruginosa. These proteins form eight transmembrane, antiparallel, amphipathic ß-strands. They form ß-barrels with short turns at the periplasmic barrel ends and long, flexible loops at the external ends (42). OmpA of E. coli is required for bacterial conjugation, maintaining outer membrane stability, and determining cell shape and ability to grow in low-osmolarity medium.

A protein of the FadL family (TC 1.B.9) is present. The E. coli FadL protein is responsible for long-chain fatty acid transport across the outer membrane. Residues involved in fatty acid binding and transport have been distinguished and identified (21).

Other transporters in this category include three autotransporters of a serine protease (TC 1.B.12). These autotransporters are virulence factors which cross the cytoplasmic membrane via the Sec (general secretory) pathway (TC 3.A.5), and following cleavage of their N-terminal targeting sequence, they enter the periplasm of the gram-negative bacterial cell envelope. The C-terminal 250 to 300 amino acyl residues fold and insert into the outer membrane to give rise to a putative ß-barrel structure with 14 transmembrane ß-strands. This structure forms a pore through which the N-terminal virulence factor is transported to the extracellular medium.

Another ß-barrel transporter involved in virulence is a toxin export channel (TC 1.B.20), which is involved in export across the outer membranes of gram-negative bacteria of various toxin proteins. For example, the ShlB toxin export channel of Serratia marcescens exports hemolysin through the outer membrane after it is secreted by the type II general secretory pathway (TC 3.A.5) system across the cytoplasmic membrane (20). These proteins are thought to form ß-barrel channels in the outer membrane through which Xylella hemolysin III toxin (XF0175) may be exported.

Certain domains of outer membrane proteins are exposed at the outer surface and therefore may be good candidates for vaccines in gram-negative bacteria. For example, porin F, which is one of the major proteins of the outer membrane of P. aeruginosa, has been identified as a candidate for a vaccine because it antigenically cross-reacts in all serotype strains of P. aeruginosa of the International Antigenic Typing Scheme (10). Some of the outer membrane channel-forming proteins of X. fastidiosa identified above may be considered targets for studies of a possible vaccine.

Pore-Forming Toxins

X. fastidiosa has four RTX toxins (TC 1.C.11), which are bacterial pore-forming exotoxins (Table 2). They are secreted from the bacteria, and after processing, they insert into the membranes of animal cells. There they cause cell rupture by mechanisms that are not well understood.

Electrochemical Potential-Driven Transporters

Xylella has 49 secondary transporters, which are listed in Table 3, that couple proton electrochemical potential of the cytoplasmic membrane to active transport events. Respiration probably provides most of the energy for generation of this proton motive force. A total of 39 major secondary transporters were identified (Table 3), including symporters, antiporters, uniporters, and the resistance-nodulation-cell division (RND) family, along with 10 other energizers (Table 3) of the TonB family.


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  		TABLE 3. Electrochemical potential-driven transportera

 
Symporters, antiporters, and uniporters.

(i) Major facilitator superfamily. The major facilitator superfamily is a large and diverse superfamily that includes several hundred sequenced members. They catalyze uniport, solute:cation (H+ or Na+) symport, and/or solute:H+ or solute:solute antiport. Just eight proteins in X. fastidiosa belong to the major facilitator superfamily (Table 3). In contrast, 64 proteins of this kind are found in E. coli. The Xylella members export drugs and import metabolites and fucose.

One interesting member of the major facilitator superfamily is involved in the uptake of oligopeptides (TC 2.A.1.25), including cell wall degradation products (peptides and glycopeptides, including N-acetylglucosaminyl-ß-1,4-anhydro-N-acetylmuramyl-tripeptide) as well as penicillin derivatives. Note also the presence in X. fastidiosa of two additional porters involved in the uptake of oligopeptides, TC 2.A.17 and 2.A.67, which are oligopeptide:H+ symporters. In this respect, it is interesting that no ABC-type active transporter of oligopeptides (TC 3.A.1.5.1) was found in X. fastidiosa. The ABC-type oligopeptide transporters also take up amino glycoside antibiotics, such as kanamycin and streptomycin, as well as cell wall-derived peptides such as murein tripeptides. The oppABCDF genes of Salmonella enterica serovar Typhimurium are well-characterized members of this family, which is represented in all 25 fully sequenced bacteria except X. fastidiosa, Neisseria meningitidis, and Rickettsia prowazekii.

(ii) Other secondary porters. The ATP/ADP translocase antiporter (XF1738) (TC 2.A.12) is the most important member in this class. It has been found only in Chlamydia and Rickettsia spp., two obligate intracellular bacterial parasites of eukaryotic cells. A homolog has been sequenced in Arabidopsis thaliana and is supposed to be localized to the intracellular plastid membrane, where it functions as an ATP importer (18). The X. fastidiosa ATP/ADP translocase is similar to the Npt1 translocase of Chlamydia trachomatis (E value, 1e-16; 22% identity, 38% similarity, and 90% coverage of the Chlamydia example). XF1738 is classified by the cluster of orthologous groups (46) analysis as belonging to COG3202, ATP/ADP translocase. The Xylella translocase has 10 TMSs, identical to the number of TMSs predicted by PSORT (25) for the Chlamydia ATP/ADP translocase.

In C. trachomatis the transporter is an exchange translocase specific for ATP and ADP (47). It functions to take up ATP from the eukaryotic cell cytoplasm into the bacterium in exchange for ADP, thus providing a source of energy for the bacteria (47). It is quite unexpected that a nonintracellular bacterium such as X. fastidiosa should have an ATP/ADP translocator, and it is tempting to speculate that X. fastidiosa may utilize the so far uncharacterized plant xylem ATP as an additional source of energy. In fact, preliminary results in our laboratory show that X. fastidiosa takes up ATP from the culture medium (T. Koide, S. L. Gomes, and S. Verjovski-Almeida, unpublished data) with kinetics very similar to that shown for the Chlamydia translocase (47).

Additional porters in X. fastidiosa include 37 proteins comprising 23 systems for export or import across the inner membrane of citrates, oligopeptides, amino acids, proteins, drugs, metals, antimicrobial agents, and ions. The most prevalent is the RND family (TC 2.A.6), with six different systems and a total of eight proteins. These porters are ubiquitously present in bacteria, archaea, and eukaryotes. Members of the RND superfamily all probably catalyze substrate efflux via an H+ antiport mechanism. The substrates are either heavy metals (e.g., Co2+, Zn2+, Cd2+, and Ni2+), multiple drugs, or proteins.

Two interesting systems deserve special mention. One of them is the type V secretion pathway or twin-arginine transporter (TC 2.A.64), which gets its name because it transports proteins characterized by a leading sequence (S/T)RRXFLK with two arginines. In E. coli this system has five components, TatABCDE, with 1, 1, 6, 0, and 1 TMSs, respectively, and sizes of 98, 171, 258, 264, and 67 amino acids, respectively, forming a gene cluster. In X. fastidiosa we found five homologs, but they are not in one-to-one correspondence with the E. coli proteins, and only three of them are clustered together. Xylella ORFs XF0564, XF0563, and XF0562, with 0, 0, and 6 TMSs, respectively, and sizes of 71, 140, and 246 amino acids, respectively, are similar to TatA, TatB, and TatC, respectively. Two homologs of TatD exist, XF0177 and XF1913, both with 0 TMSs and sizes of 260 and 268 amino acids, respectively. However, the similarity of XF1913 to TatD is much higher, and XF0177 shows higher similarity to an unrelated protein (YiiV of E. coli) than to TatD. Component TatE was not found, perhaps due to its small size. Also, TatA and TatE may have similar roles, as they can partially substitute for each other in the E. coli type V secretion pathway system. These findings support the existence of a twin-arginine transporter system in X. fastidiosa. In addition, X. fastidiosa has one protein (XF0842) with the leading signal motif TRRXFLK. In fact, XF0842 has the leading sequence 3TRRTFLR9, with a conservative replacement of R for K in the seventh position of the motif. XF0842 is similar (E score, 3e-79) to a putative secreted protein from Streptomyces coelicolor (CAB61925).

The other interesting note concerns the inorganic carbon transporter (TC 2.A.73). X. fastidiosa has an ORF with similarity to ictB, an Na+:HCO3- symporter protein so far found only in the cyanobacterium Synechocystis (3) and believed to effect transport of inorganic carbon in the form of HCO3- to be processed in photosynthesis in Synechocystis. This is surprising and suggests that X. fastidiosa may count on HCO3- from the xylem of citrus plants.

Energizers

Among its predicted genes, X. fastidiosa has three clusters and two isolated predicted genes whose translations were classified in the TonB family (TC 2.C.1). The cytoplasmic membrane protein TonB couples the proton electrochemical potential of the cytoplasmic membrane to active transport events at the outer membrane of gram-negative bacteria via outer membrane receptors (members of the outer membrane receptor family, TC 1.B.14). All the Xylella proteins hit the E. coli example systems TonB-ExbB-ExbD and TolA-TolQ-TolR. TonB and TolA are believed to serve the same function, and ExbB and TolQ are homologous, as are ExbD and TolR.

One of the Xylella clusters (XF0009, XF0010, XF0011, and XF0012) contains four sequences with high similarity to the TonB-ExbB-ExbD1-ExbD2 system of E. coli. A second cluster (XF1899 and XF1900) contains only two sequences, similar to TolQ and TolR, but lacks a TolA homolog. However, the two isolated X. fastidiosa predicted genes (XF2287 and XF2327) are both similar to TonB, and we propose that one of them could function as TolA for this cluster. Finally, the third cluster (XF1079 and XF1080) also contains two sequences, one of them similar to both ExbB and TolQ, and the other similar to both ExbD and TolR.

Primary Active Transporters

A total of 32 primary active transport systems which couple either the hydrolysis of the phosphate bond of ATP or the oxidoreduction of NADH:ubiquinone to the active transport of ions and other small molecules were identified in Xylella. Xylella primary active transporters are identified in Tables 4 and 5 below.


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  		TABLE 4. Primary active transporters: ABC transportersa

 

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  		TABLE 5. Primary active transporters other than ABC transportersa

 
Transport driven by phosphate bond hydrolysis. (i) ABC superfamily. The ATP-binding cassette (ABC) transporter superfamily are present in all forms of life and use the energy released by hydrolysis of ATP to promote active transport of substances across the cytoplasmic membrane. Some systems effect uptake while others effect export, but no known system has both functions. A typical ABC system for uptake of solutes is composed of three units appearing together as a gene cluster: a receptor, a membrane component, and a cytoplasmic, ATP-binding component. The receptors are usually not required in the ABC export systems. The best-conserved component is the ATP-binding one; even simple Blast searches made with a typical member can easily uncover all the others. This motif is quite conserved, and usually no false-positives result.

In X. fastidiosa, the ATP-binding cassette superfamily is the best represented superfamily, and we found 23 systems comprising a total of 43 proteins (Table 4). Twelve of them are predicted based solely on the presence of the ATP-binding component, and therefore the specified subfamily is tentative. In all such cases we have indicated in the tables that the membrane component is lacking, and when appropriate, we also indicate that the receptor is lacking for the putative uptake systems. Finding the specific substrates for those transporters will be an interesting subject for future study.

(a) Uptake ABC systems. Nine systems were classified as belonging to uptake families (TC 3.A.1.1 to 3.A.1.99), including members for the import of sugar (CUT1), glutamate and other polar amino acids (PAAT), hydrophobic amino acids (HAAT), sulfate (SulT), phosphate (PhoT), vitamin B12 (VB12T), nickel (PepT), and bicarbonate (NitT). The CUT1 system is well identified, although its cytoplasmic component gene is located far from the other components, which cluster together in the chromosome. The VB12T system is not well identified, with both the cytoplasmic and membrane components missing and just the receptor present. One of the PAAT systems (XF0874 and XF0875) lacks the receptor, as does the NitT system. The other putative PAAT system has four clustered genes; however, only the cytoplasmic ATP-binding component (XF0421) has homology to the glutamate porter gene gluA of Corynebacterium glutamicum, a typical example of an ABC four-member uptake porter. This ORF also has good similarity to the ttg2A gene of Pseudomonas putida, a member of the ABC family that has been implicated in toluene resistance (19) and is not listed among Saier's examples.

The remaining three ORFs in this Xylella cluster (XF0420, XF0419, and XF0418) have similarity to genes ttg2BCD, which are part of the P. putida operon involved in toluene resistance (19). It is expected that toluene resistance would be related to extrusion of toluene; however, the operon in P. putida (19) has been shown to be a four-member system typical of uptake ABC family systems. It is possible that knockout of the ttg2 genes (19) has made the otherwise toluene-resistant P. putida strain nonviable by affecting the uptake of energy fuel and not necessarily by interfering directly with the toluene resistance mechanism. In fact, the authors who described the ttg2 gene of P. putida pointed out that the ttg2 mutant is very sensitive to short-term treatment with toluene, suggesting the importance of this transporter in toluene resistance; however, as they state in their article, at present it is not clear whether this gene encodes a protein acting as a toluene pump (19). The authors suggested alternatively that the gene might encode a transporter protein functioning in outer membrane synthesis, which is an important barrier to penetration by growth inhibitors (19). It should be noted that P. putida has a ttg3 gene that codes for a toluene efflux pump, which plays an important role in P. putida toluene resistance (19), whereas X. fastidiosa does not have any gene similar to ttg3.

Further functional characterization of this putative PAAT Xylella system would clarify its possible glutamate substrate specificity as well as the direction of transport. The HAAT and PepT systems have only the cytoplasmic unit, both lacking the membrane and receptor components; specificity has been assigned based on the GenBank (http://www.ncbi.nlm.nih.gov/) best match of the cytoplasmic component, and further experimental characterization of a membrane unit for these systems would be of utmost relevance. Both SulT and PhoT are well identified, each having four clustered units, including two membrane components.

(b) Export ABC systems. The remaining 14 systems are for export. Thus, we have export of lipopolysaccharides (LPSE) and lipooligosaccharides (LOSE), drugs (DrugE1 and DrugRA1), lipids (LipidE), heme (HemeE), proteins (Prot1E and Prot2E), and sodium (NatE). The best characterized are the LPSE, DrugE1, HemeE, and NatE systems.

The ABC export systems involved in drug transport (ProtE1, ProtE2, DrugE1, and three members of DrugRA1) are probably responsible for conferring resistance to antibiotics and for excretion of antibiotics (macrolides) and toxins.

It is noteworthy that X. fastidiosa has a sodium export system (NatE) that is similar to the NatAB system of Bacillus subtilis, in which the extrusion of sodium has been very well characterized (6). Sodium extrusion via an ABC transport system is found in all eight fully sequenced archaea except Thermoplasma acidophilum. Among the 24 fully sequenced eubacteria, only two gram-positive ones, B. subtilis and D. radiodurans, have such sodium extrusion ABC transporter, along with Thermotoga maritima, a hyperthermophilic bacterium that is closely related to the low-G+C gram-positive bacteria (16). In T. maritima it has been proposed that there has been extensive lateral gene transfer with archaea (26) as part of the mechanism of evolution towards thermophilicity. It will be interesting to study the physiology of sodium ion transport in X. fastidiosa to understand the role of such a sodium transport system in adapting X. fastidiosa to the plant xylem.

Four of the export systems (LPSE, HemeE, DrugE1, and NatE) exhibit their components in gene clusters and have a membrane as well as a cytoplasmic component. The other 10 have only the cytoplasmic unit, and no gene in the vicinity is related to the ABC superfamily. Again, experimental confirmation of the specific substrates that are exported by those transporters will be an interesting subject for future study.

(ii) Other phosphate bond-driven transporters. Seven systems comprising 30 proteins make up the rest of the phosphate bond hydrolysis-driven transporters (Table 5). X. fastidiosa has an F-ATPase (TC 3.A.2.1.1) with eight subunits, very similar to E. coli and other bacterial systems. This F-ATPase or ATP synthase system is complete in X. fastidiosa, indicating that ATP synthesis is driven by a chemiosmotic proton gradient.

It is interesting that no P-type cation transport ATPase (TC 3.A.3) was found in X. fastidiosa. The P-type ATPase family of ion transport proteins is a very well characterized system, and the transport mechanism involves phosphorylation (by the gamma-phosphate of ATP) of the aspartyl residue in the conserved motif DKTGT(L/I)T during the catalytic and transport cycle (17). All fully sequenced archaea, bacteria, and eukaryotes have P-type ATPases which transport protons, sodium, potassium, and other ions at the expense of ATP. There are four exceptions: Pyrococcus horikoshii is the only archaea among the eight fully sequenced that does not have a P-type ATPase. Among the 24 fully sequenced eubacteria, only R. prowazekii, Borrelia burgdorferi, and X. fastidiosa do not have a P-type cation transport ATPase.

It is interesting that the three eubacteria have in common the fact that they are not free-living organisms (X. fastidiosa lives in the plant xylem, B. burgdorferi is a blood parasite, and R. prowazekii is an intracellular parasite) that are transmitted by insect vectors. It is tempting to speculate that transmission via an insect vector has obviated the need for this ATP-driven cation transport. In turn, it is possible that this adaptation has caused X. fastidiosa to have a distinct way of regulating its cation concentration. Characterization of ion transport regulation in X. fastidiosa should be an interesting subject for study, since this might be a special target for controlling and interfering with its growth.

A type II or general secretory pathway (TC 3.A.5) is complete. Notice that in our tables, proteins SecD and SecF (XF0225 and XF0226, respectively) are present as examples both in this family and in their own specific family as secondary transporters (TC 2.A.6.4, SecDF), and YajC (XF0224) is also cross-listed here and in the SecDF-associated single transmembrane protein family (TC 9.B.18).

It should be emphasized that no type III (virulence-related) secretory pathway system (TC 3.A.6) was found in X. fastidiosa. The type III system is responsible for injection of virulence-related bacterial proteins directly into the cytoplasm of host cells (13, 49). In bacterial plant pathogens, type III secretory systems are responsible for injecting the so-called avirulence (avr) genes into host plant cells (1), which in turn elicits plant responses that will ultimately result in a limited host range, often confined to members of a single plant species or genus. In accordance with the absence of a type III secretory system, no genes with similarity to the known avirulence genes were found in the Xylella genome. It is apparent that these genes are not required for Xylella-host interactions, and it is possible that the insect-mediated mechanism of transmission and vascular restriction of the bacterium obviates the necessity of host cell infection.

A type IV (conjugal DNA-protein transfer or VirB) secretion pathway (TC 3.A.7) is found on the large plasmid with one duplicated gene (virB10, XF2049) in the main chromosome. The type IV secretion pathway system is a multisubunit protein complex that spans the two membranes of the gram-negative bacterial cell envelope and exports DNA-protein complexes out of the cell and into the cytoplasm of a recipient cell, which can be a bacterial, yeast, or plant cell. The VirB system of agrobacterial species is the typical type IV system and is specifically designed to transfer T-DNA (transferred DNA) into plant cells (7). X. fastidiosa has all six protein homologs of the VirB system (VirB4 and VirB11 are ATPases, VirB6 and VirB10 form an integral membrane transport pore, and VirB7 and VirB9 form an outer membrane complex) which comprise the minimum structural and catalytic elements of the dual-membrane channel complex. In fact, VirB7 was not detected by Glimmer (a gene prediction software tool) and was not present in the original annotation of the genome (44), being recently identified by a detailed search against members of the VirB family by Marques et al. (23).

Although members of the type IV secretion family share many characteristics, not all systems contain the same sets of genes. Thus, additional members of the VirB family such as VirB3 and VirB8 are found in X. fastidiosa, while VirB2 and VirB5 are not readily identifiable by similarity. A thorough description of this Xylella conjugative system has been reported by Marques et al. (23). Given the conservation between the genes involved in the conjugation process and in the processes of pathogen-host DNA transfer mediated by the VirB system, it is possible that a type IV pathway has a role in X. fastidiosa pathogenesis (23).

X. fastidiosa has a bacterial competence-related DNA transformation transporter (TC 3.A.11), a system that is found in many gram-negative as well as gram-positive bacteria and confers natural competence, i.e., the bacteria are able to take up DNA under normal physiological conditions. DNA binds to the cell surface via a type IV pilus; the DNA is usually cleaved, generating double-strand breaks, and one of the two DNA strands is taken up while the other strand is degraded on the external surface of the cell (9). The best-characterized uptake system is that in B. subtilis, in which a DNA-binding receptor protein, ComEA, on the external surface extracts the DNA molecule from the type IV pilus and feeds it into the transmembrane channel protein, ComEC, that spans the membrane 10 times (9). Both ComEA and ComEC are required for DNA transport into the cytosol, and similar proteins were found in X. fastidiosa (XF0593 and XF1078, respectively). In B. subtilis, ComFA is an ATP-driven DNA translocase with homology to E. coli DNA/RNA helicases. The ComEA-ComEC-ComFA complex probably drives single-stranded DNA through the ComEC channel in a process energized by ComFA-catalyzed ATP hydrolysis (9). Again, an ATP-dependent RNA helicase protein similar to ComFA was found in X. fastidiosa (XF0252).

The fimbrilin/protein exporter family (TC 3.A.12) is present in two copies in the Xylella genome. Each of the exporters in the fimbrilin/protein exporter family consists of two members, an integral membrane protein having three putative TMSs and an ATPase localized on the cytoplasmic side of the membrane (11). Both copies are complete in the Xylella genome, being similar to the pilin secretion/fimbrial assembly system PilBC of P. aeruginosa. These are part of the general secretory pathway (TC 3.A.5) of gram-negative bacteria.

Oxidoreduction-driven transporters. X. fastidiosa has a complete proton ion-translocating NADH dehydrogenase I system (Table 5), which is a multisubunit enzyme that couples electron transfer from NADH to ubiquinone with proton translocation from the negative inner to the positive outer side of the bacterial membrane (12). Fourteen units make up the complete NADH dehydrogenase I complex (TC 3.D.1), similar to the one found in E. coli (also called NADH:ubiquinone oxidoreductase), which extrudes H+ (12). Three other proteins compose a complete quinol:cytochrome c reductase system (TC 3.D.3), for which we were unable to pinpoint the subfamily. Two of the components (cytochrome b and the Rieske Fe2S2 protein) are similar to the TC 3.D.3.1 Paracoccus denitrificans examples, while the third one (cytochrome c1) is similar to a TC 3.D.2.1 Bos taurus example. The quinol:cytochrome c reductase system transfers electrons from a quinol to cytochrome c and links this electron transfer to proton extrusion (4). The three subunits of a proton-translocating cytochrome oxidase system (TC 3.D.4) are also present. This multisubunit enzyme reduces O2 to water and concomitantly pumps four protons across the membrane (24).

The three oxidoreduction-driven transport systems described above provide X. fastidiosa with a complete respiratory chain that generates an electrochemical proton motive force able to drive ATP synthesis through the F-ATPase or ATP synthase (TC 3.A.2.1.1) complex.

Phosphotransferase System

The phosphotransferase system (PTS) has been described in several bacteria and is used for import of carbohydrates into the cell (36, 37). Sugar molecules are phosphorylated upon transport to keep them inside the cell. The system involves two auxiliary enzymes, which interact with phosphoenolpyruvate, from which they acquire both the energy for transport and the phosphate group that will be transferred to the imported substrate. These auxiliary enzymes have to interact with the permease component, located in the membrane.

The availability of such a system for sugar transport in X. fastidiosa remains a mystery. On one hand, both auxiliary enzymes the PTS enzyme I (8.A.7) and the PTS HPr protein (8.A.8) are present in a cluster (XF1402 and XF1403) (Table 6). On the other hand, no permeases have been found. The best candidate in the entire genome is neighboring ORF XF1404, which is weakly similar (e-value, 7e-05; 22% identity, 44% similarity, and 37% coverage) to a mannose permease IIAB component (P08186) of one of the E. coli PTSs, the mannose (glucose, glucosamine, and fructose) porter (TC 4.A.6.1.1). However, the other subunits were not found, especially PTS IIC, which is presumed to be the sugar-transporting channel component, and it is doubtful whether just one subunit could make a functioning permease.


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  		TABLE 6. PTS and auxiliary transport proteinsa

 
It might be that a new subfamily of permeases, not previously described, exists in X. fastidiosa. It is noteworthy that Treponema pallidum, Chlamydia trachomatis, and Neisseria meningitidis retain only a few of the PTS genes and lack a typical PTS permease channel component; the system could play a solely regulatory role in sugar and nitrogen metabolic pathways (41). In fact, the PTS has been shown to participate in multiple physiological control processes that regulate sugar metabolism in bacteria (37, 40, 43, 45). In E. coli and many other gram-negative bacteria, regulation is mediated by HPr and enzyme IIAGlc (40) and by the PTS pathway of the Ntr type comprised of enzyme I(Ntr) (35) and enzyme IIANtr (5, 33). X. fastidiosa possesses neither EIIAGlc nor EIIANtr; in fact, among the 22 fully sequenced bacteria having an HPr-related protein, X. fastidiosa and Ureaplasma urealyticum are the only ones that do not possess EIIANtr. Instead, we found that X. fastidiosa has a mannose-specific enzyme IIA component (TC 4.A.6.1.1), and it should be of interest to study the eventual role of this PTS component in sugar metabolism control in X. fastidiosa.

Yet another neighboring protein, XF1406, is an HPr kinase/phosphatase, known to phosphorylate and dephosphorylate Ser-45 of the phosphoryl carrier HPr protein, which again suggests that a functional, not yet completely identified sugar phosphotransferase system might be present in X. fastidiosa.

Auxiliary Proteins

The majority of the auxiliary proteins listed in Table 6 were already mentioned earlier in connection with the system that they support. The only exception is a ß-subunit of voltage-gated ion channels (TC 8.A.5), which conforms to the existence of a protein (TC 1.A.1) in X. fastidiosa, a potassium channel. Potassium channels in this family are formed by four identical {alpha}-subunits, and in some cases also four oxidoreductase ß-subunits that co assemble with the tetramer and remain tightly adherent to it. The X. fastidiosa voltage-gated ion channel protein is similar to KscA, a Streptomyces lividans K+ channel. The three-dimensional structure of KscA has been solved, and the protein appears as a four-unit tetramer only, without ß-subunits (8). Rat K+ channel structure has recently been resolved, and an {alpha}4-ß4 complex was determined (14), but the biological function of the oxidoreductase ß-subunits of K+ channels remains unsolved. It is not clear at this point what would be the mechanism of interaction and the function of the X. fastidiosa channel ß-subunit and its voltage-gated ion channel protein.

Unknown and Uncharacterized Transporters

Eighteen transporters are similar to examples from the TC 9.A (unknown transporters) and 9.B (uncharacterized transporters) families (Table 7 particular cases here. One is the pair XF1400-XF1401, both of which partially match an example in the MgtE family (TC 9.A.19). The matches occur in different parts of the example, and together the two ORFs, which are in two different frames in the genome, almost entirely cover the MgtE sequence. It may be the case either that in X. fastidiosa the function of this transporter is performed by two proteins or that the mgtE gene is inactive in X. fastidiosa because of a frameshift truncation.

The other case is the MarC homolog. In both E. coli and S. enterica serovar Typhimurium, this gene is encoded in a transcriptional unit at one side of the marO operator. On the other side, a divergently positioned transcriptional unit encoding the marRAB operon is found. None of the other mar genes were found in X. fastidiosa.

In addition to the 18 proteins described above, which are classified in Saier's transport protein database, we have identified another 55 proteins that do not match any example in the database and have four or more TMSs (Table 7). They were classified either as hypothetical transport proteins (when they did not match any other GenBank protein or matched proteins classified as hypothetical) or as undefined (when they matched GenBank proteins with defined names but not well-defined function) (Table 7). Many of these Xylella proteins are classified in the COG database (46), and upon inspection of the assignments, it can be seen that a good number of them are reported as predicted permeases or as uncharacterized membrane-associated proteins (Table 7). We suggest that these proteins are good candidates for exploratory experimentation on the ability of solutes to move across the Xylella membrane.


   CONCLUSIONS
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 References
 
The detailed analysis of transport systems described here uncovered important characteristics of X. fastidiosa, including hints that can lead to control of the disease caused by this bacterium on citrus trees. Our present work confirmed for the most part the observations made in the sequencing paper (44), but also pointed out a few places where an alternative interpretation of the data is possible, as indicated below. The comparison with other bacterial genomes, some of them available only recently, suggested correlations between the absence of certain transport systems and the life cycle of the parasite.

The sequencing paper (44) mentions a total of 140 proteins involved in transport (4.8% of all ORFs); the present analysis has classified 69 new transporters and suggests that in X. fastidiosa there are at least 209 transport proteins, making up 7.4% of all ORFs. In addition, more transporters could eventually be found by experimentation focused on the 55 Xylella proteins with four or more TMSs that do not match any known transport protein example. Considering the major transporters computed by Paulsen et al. (31), X. fastidiosa has only 2.7% of all ORFs identifiable as major transporters, which makes it the eubacterium with the lowest percentage of ORFs involved in transport, closer to two archaea, M. jannaschii (2.4%) and M. thermoautotrophicum (2.4%) (31). Also, X. fastidiosa exhibits a predominance of secondary transporters over primary ones, following an apparently general tendency of aerobic prokaryotes.

Regarding ways of controlling the disease, one possibility is a vaccine. Protein XF0343 (TC 1.B.6) has long, flexible loops exposed on the exterior of the cell and is a good antigen candidate. Other possibilities should arise from the answer to the question of which compounds of the xylem sap are imported by X. fastidiosa. Previous analysis (44) suggested that glycerol, certain amino acids, and possibly cellulose should be among the compounds imported. The present study adds ATP and inorganic carbon to the list of possible imported compounds. It is interesting that the bacterium apparently depends strictly on carbohydrates, both as an energy source and as anabolic precursors.

The presence of an ATP/ADP translocator is especially intriguing, since the only other organisms in which it has been found are obligate intracellular parasites. Could it be that X. fastidiosa infects plant cells as well as the xylem?

The lack of a P-type ATPase is also surprising. Only two other completely sequenced eubacteria lack this cation transport system, and they have in common with X. fastidiosa the fact that all are parasites transmitted by insects. It would be interesting to construct a genetically modified X. fastidiosa with, say, a potassium uptake P-type ATPase from E. coli and observe the effects on growth speed and interaction with the insect host.

We confirmed by renewed searches the puzzling fact that X. fastidiosa has all the apparatus of a PTS except permease components IIB and IIC. If the system is functional for sugar transport, these proteins cannot be missing. The most plausible explanation seems to be that a new family of permeases, not yet characterized as such, is to be found among the hypothetical proteins. Alternatively, PTS might play a solely regulatory role in sugar metabolism.

Finally, our analysis suggests an alternative functional assignment for the ttg2 cluster of genes, found both in X. fastidiosa and in P. putida, in which it was proposed to be either a toluene exporter or a transporter indirectly involved in toluene resistance (19). The presence of a receptor in the X. fastidiosa ABC system implies that it is an import rather than an export system. Based on the similarity of its cytoplasmic component, we tentatively placed it in the PAAT family as a glutamate importer. The fact that its knockout impaired P. putida's intrinsic resistance to toluene may be due to impaired synthesis of cell wall components (19) or to the lack of the extra energy supply that would be available from glutamate.


   ACKNOWLEDGMENTS
 
We acknowledge support from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) for supporting all X. fastidiosa sequencing work in our laboratories and the analysis work shown here.


   FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, São Paulo, SP 05508-900, Brazil. Phone: 55-11-3091-2173. Fax: 55-11-3091-2186. E-mail: verjo{at}iq.usp.br. Back


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