# Transcriptome Analysis of the PhytobacteriumXylella fastidiosa Growing under Xylem-Based Chemical Conditions

Authors: Maristela Boaceff  Ciraulo; Daiene Souza Santos; Ana Claudia de Freitas Oliveira Rodrigues; Marcus Vinícius de Oliveira; Tiago Rodrigues; Regina Costa de Oliveira; Luiz R. Nunes
Journal: Journal of Biomedicine and Biotechnology
 (2010)

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## Abstract

Xylella fastidiosa is a xylem-limited bacterium responsible for important plant diseases, like citrus-variegated chlorosis (CVC) and grapevine Pierce's disease (PD). Interestingly, in vitro growth of X. fastidiosa in chemically defined media that resemble xylem fluid has been achieved, allowing studies of metabolic processes used by xylem-dwelling bacteria to thrive in such nutrient-poor conditions. Thus, we performed microarray hybridizations to compare transcriptomes of X. fastidiosa cells grown in 3G10-R, a medium that resembles grape sap, and in Periwinkle Wilt (PW), the complex medium traditionally used to cultivate X. fastidiosa. We identified 299 transcripts modulated in response to growth in these media. Some 3G10R-overexpressed genes have been shown to be upregulated in cells directly isolated from infected plants and may be involved in plant colonization, virulence and environmental competition. In contrast, cells cultivated in PW show a metabolic switch associated with increased aerobic respiration and enhanced bacterial growth rates.


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## Body

## 1. Introduction

The phytobacteriumXylella fastidiosa was described by Wells et al. [1] and has been found to be associated with the development of a wide variety of plant diseases, such as Citrus-Variegated Chlorosis (CVC) in orange trees, Pierce’s disease (PD) in vineyards, Phony Peach disease (PP), Periwinkle Wilt and leaf scorch diseases in plum, elm, maple, pecan, oak, sycamore, and coffee ([2, 3], reviewed in [4]). Due to the presence of economically important crops in this list, X. fastidiosa has been the subject of intensive research over the past years [5, 6] and the genome sequencing of four different strains has been accomplished: the 9a5c isolate (causative agent of CVC) was the first phytopathogenic bacterium completely sequenced in 2000 [7]. A few years later, two strains isolated from oleander and almond trees had their genomes partially sequenced and annotated [8]. Finally, a fourth strain, Temecula 1, isolated from grapevines and responsible for PD in California has also been sequenced to completion [9].The elucidation of the complete genomic sequence ofX. fastidiosa strains was followed by an extensive in silico evaluation of the bacterium’s presumed proteome, allowing the formulation of a virtual metabolome that provided a comprehensive view of the major biochemical processes that occur in this microorganism [7]. Nonetheless, the exact mechanism(s) involved in the process of host infection and colonization, as well as with the onset of CVC, are yet to be identified and characterized in the X. fastidiosa genome [7]. Important information regarding the functionality of different gene products and pathogenicity mechanisms in X. fastidiosa could be obtained through the evaluation of differential gene expression using cells submitted to variable culturing conditions, especially those that resemble the environment found inside the plant. Xylem-inhabiting microorganisms normally display a fastidious nature and cannot be cultured in conventional bacteriological media. Thus, a series of specially formulated media were developed for their axenic cultivation. The most widely employed, such as PD2 [10], PW [11], SPW [12], PYE, GYE [13] and BCYE [14], are complex media, which include peptone, tryptone, soytone, and yeast extract from various sources, as well as hemin chloride or ferric pyrophosphate (as iron sources), aminoacids, inorganic salts, citrate, succinate, starch, BSA, or activated charcoal. However, given the general characteristics of plant sap, xylem-dwelling endophytes are likely to thrive in nutrient-limiting conditions and must be able to adapt accordingly [15]. A few years ago, Leite et al. [16] have described the development of a xylem-based, chemically defined medium (called 3G10R), which supports in vitro growth of X. fastidiosa strains. Moreover, X. fastidiosa cells grown in this medium present some important characteristics that may be associated with colonization and pathogenicity, such as increased aggregation capacity and biofilm formation. This medium provided a new tool that may allow the in vitro study of some important characteristics presented by the bacteria during the infection process in planta.Thus, we have employed competitive hybridizations on microarrays to evaluate the global transcriptional profile ofX. fastidiosa cells grown in 3G10R, when compared to cells grown in PW, the standard complex medium used to cultivate this bacterium under laboratory conditions. These experiments allowed the identification of 299 genes that displayed statistically significant transcription modulation in response to growth in the two media. Some 3G10R-upregulated genes had their expression profiles confirmed by Real-Time qPCR and are likely to be relevant to bacterial adaptation to the plant xylem, such as adhesion to the substrate and competition with other microorganisms. Incidentally, independent studies have confirmed the specific upregulation of some of these genes in X. fastidiosa cells that display increased infective capacity and in bacteria directly isolated from plants, reinforcing the idea that the chemical characteristics of 3G10R are likely to induce genes that are naturally expressed by X. fastidiosa during the process of xylem colonization [17]. Other transcriptional alterations seem to correlate with significant changes in the cell’s overall energetic metabolism and growth rate, as a reduction in the respiratory activity is observed when cells are grown in 3G10R.


## 2. Materials and Methods

### 2.1. Culturing X.  fastidiosa Cells

PW and 3G10R liquid media have been prepared essentially as described by Davis et al. [11] and Leite et al. [16], respectively. Cells of X. fastidiosa 9a5c have been routinely kept in our laboratory, for over a year, in 20 ml of liquid cultures, which were incubated in an orbital shaker at 28°C and 100 rpm. One-milliliter (1 ml) aliquots were transferred to 19 ml of fresh media every 4-5 days.To evaluate the behavior ofX. fastidiosa cells under xylem-based chemistry conditions, bacterial cultures were grown in PW for 3 days, until an OD600 = 0.25 (late phase of exponential growth) was reached. A one milliliter-aliquot (1 ml) of this culture was used to inoculate 19 ml of liquid 3G10R and PW media. Bacterial growth in both cultures was monitored on a daily basis, through OD600 measurements, providing a direct comparison between X. fastidiosa growth patterns observed in 3G10R and standard PW medium.


### 2.2. Microarray Fabrication

X. fastidiosa microarrays have been constructed as previously described [18, 19].  Representative sequences from approximately 2200 ORFs from the X. fastidiosa genome (>90% coverage) were PCR amplified, purified, and spotted onto CMT-GAPS silane-coated slides (Corning), using an Affymetrix 427 arrayer, according to the manufacturer’s instructions.


### 2.3. RNA Extraction, cDNA Labeling, and Hybridization

To evaluate and compare the bacterial transcriptome profiles in these two media, 200-ml bacterial cultures were prepared as described above and cells were harvested for total RNA extraction at day 3 (PW) and day 13 (3G10R), which allowed us to compare bacterial cultures at their maximum growth rates. The RNA samples were extracted and purified with aid of the RNAeasy kit (Qiagen), labeled by incorporation of Cy3- or Cy5-dCTP and hybridized to the microarrays, as previously described [18, 19].


### 2.4. Image Acquisition and Analysis

Images were analyzed with the TIGR Spotfinder program (v.2.2.4). All spots with median values lower than the median local background plus two Standard Deviations have been flagged and excluded from further analyses. Replicated experiments were performed with two independent RNA preparations from cells cultivated in each medium. For each pair of RNA preparations, two independent hybridizations were performed, with dye swaps within each pair. Since each microarray carries two complete copies of theX. fastidiosa genome, replicated hybridizations resulted in a series of 8 independent readings for each probe spotted in the microarrays.The results from each hybridization were submitted to a series of mathematical transformations with the aid of the software TIGR MIDAS v.2.19. These included filtering out all spots whose integrated intensities were below 10,000 a/d units, normalization between the two channels with the aid of the Lowess algorithm and SD regularization of the Cy5/Cy3 ratios across all sectors (blocks) of the array. Finally, the results from each individual experiment were loaded into the software TIGR Multi-Experiment Viewer (TMEV), v.3.01. Experiments were then normalized and genes that displayed statistically significant modulation were identified with the aid of the one-class mode of the Significance Analysis of Microarrays (SAMs) test, described by Tusher et al. [20]. The δ factor of the SAM test was adjusted to 0.69, resulting in a Median False Discovery Rate (FDR) = 0.163. For details regarding the use of the TIGR microarray software suite (TM4), see Saeed et al. [21]. Raw and normalized data from all microarray hybridizations, as well as the microarray complete annotation file have been submitted to NCBI’s Gene Expression Omnibus (GEO) and can be accessed through Series number GSE 6619. A Tab-delimited file containing the Significant Genes List and their mean expression ratios can also be accessed through this GEO Series number.


### 2.5. Real-Time qPCR

All the Real-Time qPCR and RT-PCR reactions were performed using an ABI Prism 7500 Sequence Detection System (Applied Biosystem, USA). Taq-Man EZ RT-PCR kits (Applied Biosystems, USA) were used for RT-PCR reactions, according to the manufacturer’s instructions, using 2–5μg of total X. fastidiosa RNA and 1 μl of random nonamers (4 μg/μl). The thermocycling conditions comprised an initial step at 50°C for 2 minutes, followed by 30 minutes at 60°C for reverse transcription. Taq-Man PCR Reagent kits then were used for PCR reactions using 100–200 ng of the resulting cDNA. The thermocycling conditions comprised an initial step at 50°C for 2 minutes, followed by 10 minutes at 95°C, and 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. ORF Xf1311, which encodes a rod-shaped determining protein (MreD) has been used as an endogenous control for experimental normalization, since the microarray hybridization experiments showed that this ORF is constitutively expressed in both PW and 3G10R. Primers and probes were synthesized through the Applied Biosystems Assay-by-Design service and all reactions were prepared essentially as recommended by the manufacturer.


### 2.6. Evaluation of Respiratory Rates

X. fastidiosa cells were grown into middle exponential phase in PW and subsequently transferred (in a 1 : 20 proportion) into fresh PW and 3G10R cultures. Bacterial growth in both cultures was monitored through OD600 measurements until both cultures reached stationary phase. Aliquots were taken from each culture to evaluate O2  consumption on a daily basis, until day 7 (in PW) and day 13 (in 3G10R). We defined the respiratory rate for each culture as the ratio between O2 consumption rate (ΔO2/Δmin) and the respective OD600 value obtained at each time point.Oxymetric measurements were monitored polarographically by an oxygraph equipped with a Clark-type oxygen electrode (Gilson Medical Electronics, Middleton, WI, USA) in intact cells. After measurement of the optical density, 2.0 ml of PW or 3G10R media containing bacteria were incubated at 30°C and the state 4 respiration was initiated by addition of 10 mM malate plus 10 mM glutamate. Basal respiratory rates were calculated byΔO2/Δmin ratio and the values were normalized by the optical density values.


## 2.1. Culturing X.  fastidiosa Cells

PW and 3G10R liquid media have been prepared essentially as described by Davis et al. [11] and Leite et al. [16], respectively. Cells of X. fastidiosa 9a5c have been routinely kept in our laboratory, for over a year, in 20 ml of liquid cultures, which were incubated in an orbital shaker at 28°C and 100 rpm. One-milliliter (1 ml) aliquots were transferred to 19 ml of fresh media every 4-5 days.To evaluate the behavior ofX. fastidiosa cells under xylem-based chemistry conditions, bacterial cultures were grown in PW for 3 days, until an OD600 = 0.25 (late phase of exponential growth) was reached. A one milliliter-aliquot (1 ml) of this culture was used to inoculate 19 ml of liquid 3G10R and PW media. Bacterial growth in both cultures was monitored on a daily basis, through OD600 measurements, providing a direct comparison between X. fastidiosa growth patterns observed in 3G10R and standard PW medium.


## 2.2. Microarray Fabrication

X. fastidiosa microarrays have been constructed as previously described [18, 19].  Representative sequences from approximately 2200 ORFs from the X. fastidiosa genome (>90% coverage) were PCR amplified, purified, and spotted onto CMT-GAPS silane-coated slides (Corning), using an Affymetrix 427 arrayer, according to the manufacturer’s instructions.


## 2.3. RNA Extraction, cDNA Labeling, and Hybridization

To evaluate and compare the bacterial transcriptome profiles in these two media, 200-ml bacterial cultures were prepared as described above and cells were harvested for total RNA extraction at day 3 (PW) and day 13 (3G10R), which allowed us to compare bacterial cultures at their maximum growth rates. The RNA samples were extracted and purified with aid of the RNAeasy kit (Qiagen), labeled by incorporation of Cy3- or Cy5-dCTP and hybridized to the microarrays, as previously described [18, 19].


## 2.4. Image Acquisition and Analysis

Images were analyzed with the TIGR Spotfinder program (v.2.2.4). All spots with median values lower than the median local background plus two Standard Deviations have been flagged and excluded from further analyses. Replicated experiments were performed with two independent RNA preparations from cells cultivated in each medium. For each pair of RNA preparations, two independent hybridizations were performed, with dye swaps within each pair. Since each microarray carries two complete copies of theX. fastidiosa genome, replicated hybridizations resulted in a series of 8 independent readings for each probe spotted in the microarrays.The results from each hybridization were submitted to a series of mathematical transformations with the aid of the software TIGR MIDAS v.2.19. These included filtering out all spots whose integrated intensities were below 10,000 a/d units, normalization between the two channels with the aid of the Lowess algorithm and SD regularization of the Cy5/Cy3 ratios across all sectors (blocks) of the array. Finally, the results from each individual experiment were loaded into the software TIGR Multi-Experiment Viewer (TMEV), v.3.01. Experiments were then normalized and genes that displayed statistically significant modulation were identified with the aid of the one-class mode of the Significance Analysis of Microarrays (SAMs) test, described by Tusher et al. [20]. The δ factor of the SAM test was adjusted to 0.69, resulting in a Median False Discovery Rate (FDR) = 0.163. For details regarding the use of the TIGR microarray software suite (TM4), see Saeed et al. [21]. Raw and normalized data from all microarray hybridizations, as well as the microarray complete annotation file have been submitted to NCBI’s Gene Expression Omnibus (GEO) and can be accessed through Series number GSE 6619. A Tab-delimited file containing the Significant Genes List and their mean expression ratios can also be accessed through this GEO Series number.


## 2.5. Real-Time qPCR

All the Real-Time qPCR and RT-PCR reactions were performed using an ABI Prism 7500 Sequence Detection System (Applied Biosystem, USA). Taq-Man EZ RT-PCR kits (Applied Biosystems, USA) were used for RT-PCR reactions, according to the manufacturer’s instructions, using 2–5μg of total X. fastidiosa RNA and 1 μl of random nonamers (4 μg/μl). The thermocycling conditions comprised an initial step at 50°C for 2 minutes, followed by 30 minutes at 60°C for reverse transcription. Taq-Man PCR Reagent kits then were used for PCR reactions using 100–200 ng of the resulting cDNA. The thermocycling conditions comprised an initial step at 50°C for 2 minutes, followed by 10 minutes at 95°C, and 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. ORF Xf1311, which encodes a rod-shaped determining protein (MreD) has been used as an endogenous control for experimental normalization, since the microarray hybridization experiments showed that this ORF is constitutively expressed in both PW and 3G10R. Primers and probes were synthesized through the Applied Biosystems Assay-by-Design service and all reactions were prepared essentially as recommended by the manufacturer.


## 2.6. Evaluation of Respiratory Rates

X. fastidiosa cells were grown into middle exponential phase in PW and subsequently transferred (in a 1 : 20 proportion) into fresh PW and 3G10R cultures. Bacterial growth in both cultures was monitored through OD600 measurements until both cultures reached stationary phase. Aliquots were taken from each culture to evaluate O2  consumption on a daily basis, until day 7 (in PW) and day 13 (in 3G10R). We defined the respiratory rate for each culture as the ratio between O2 consumption rate (ΔO2/Δmin) and the respective OD600 value obtained at each time point.Oxymetric measurements were monitored polarographically by an oxygraph equipped with a Clark-type oxygen electrode (Gilson Medical Electronics, Middleton, WI, USA) in intact cells. After measurement of the optical density, 2.0 ml of PW or 3G10R media containing bacteria were incubated at 30°C and the state 4 respiration was initiated by addition of 10 mM malate plus 10 mM glutamate. Basal respiratory rates were calculated byΔO2/Δmin ratio and the values were normalized by the optical density values.


## 3. Results

### 3.1. X. fastidiosa Cells Growing in PW and 3G10R Display Distinct Growth Patterns and Different Transcriptome Profiles

To evaluate the behavior ofX. fastidiosa cells under xylem-based chemistry conditions, bacterial cultures were monitored in both 3G10R and PW, the complex medium traditionally used to cultivate this bacterium in the laboratory. As observed in Figure 1, PW cultures reached higher cellular densities (OD600~0.3) in a shorter period of time (4 days) when compared to cells grown in 3G10R, which had to be cultivated for a period of 14 days in order to reach a similar cellular density (OD600~0.25). Moreover, although 3G10R cultures exhibited continuous growth over the course of the experiment, they failed to display the typical profile of a bacterial growth curve, as observed in PW cultures. Such lack of an exponential growth phase in 3G10R cultures is typically observed in bacteria growing in nutrient-restricted environments, a situation that is likely to resemble xylem conditions [22–26]. Recently, Zaini et al. [27] showed that X. fastidiosa cells grown in pure xylem sap rapidly reach stationary phase without a detectable exponential growth, probably due to nutrient limitation.Figure 1
Xylella  fastidiosa growth patterns in PW and 3G10R media. Both cultures have been made with a 1 : 20 ml inoculum of X. fastidiosa 9a5c cells grown into late exponential phase in PW (OD600 = 0.25). Cultures were then incubated in an orbital shaker at 28°C and 100 rpm. One milliliter (1 ml) aliquots were taken from each culture, on a daily basis, to monitor bacterial growth through OD600 readings. Measurements were performed in triplicate and graphic shows the average values and their respective standard deviations.To evaluate and compare the bacterial transcriptome profiles in PW and 3G10R, samples from the resulting RNAs were used in competitive hybridizations againstX. fastidiosa microarrays, as described by Nunes et al. [19]. Replicated experiments were performed with two independent RNA preparations from cells cultivated in each medium, which resulted in a series of 8 independent readings for each probe spotted in the microarrays, as described in the materials and methods. Statistical analysis of such results revealed a total of 132 genes that displayed overexpression in cells grown in 3G10R, while 167 genes were upregulated in cells grown in PW. These genes, as well as their respective changes in expression ratio are shown in Table 1. More detailed information about these genes can be obtained through the Gene expression Omnibus (GEO) webpage, through Series number GSE 6619 (see http://ncbi.nlm.nih.gov/geo). In order to access the overall reliability of these data, we have confirmed gene expression variation of several genes using an alternative approach. Thus, we performed Real-Time qPCR experiments with the same RNA samples used in the microarray hybridizations, aiming at double-checking the changes in expression of 16 genes present in Table 1 (~5% of all modulated genes). These genes have been randomly chosen from different functional categories and all displayed average expression ratios that correlate with the microarray results (see Figure 2).Table 1
List of genes that displayed statistically significant variation in gene expression. Genes with positive Log2 ratio are overexpressed in 3G10R, while genes with negative Log2 ratio are overexpressed in PW.
Functional GroupORFGeneGene ProductLog2NumberName(3G10R/PW)Intermediary MetabolismEnergy metabolism, carbon—Aerobic respirationXf0308nuoDNADH-ubiquinone oxidoreductase, NQO4 subunit-0.93Xf0310nuoFNADH-ubiquinone oxidoreductase, NQO1 subunit-1.08Xf0311nuoGNADH-ubiquinone oxidoreductase, NQO3 subunit-1.14Xf0317nuoMNADH-ubiquinone oxidoreductase, NQO13 subunit-1.03Xf0347dld1D-Lactate dehydrogenase1.18Energy metabolism, carbon—GlycolysisXf0303tpiA OR tpiTriosephosphate isomerase-0.89Energy metabolism, carbon—TCA cycleXf2548sucDSuccinyl-CoA synthetase, alpha subunit-1.67Xf1554fumCFumarate hydratase-1.47Xf1554fumCFumarate hydratase-1.36Energy metabolism, carbon—Electron TransportXf1990yneNThioredoxin-1.14Xf0620dsbDc-Type cytochrome biogenesis protein (Copper Tolerance)-0.83Degradation—Degradation of Small MoleculesXf1250rocFArginine deaminase-2.00Xf1740yliIGlucose dehydrogenase B1.45Xf2395axeAAcetylxylan esterase1.75Xf2432gtaBUTP-glucose-1-phosphate uridylyl transferase-1.14Xf0610galEUDP-glucose 4-epimerase-1.44Xf2210Dioxygenase1.00Regulatory FunctionsXf1354yybATranscriptional regulator (MARR Family)1.27Xf1354yybATranscriptional regulator (MARR Family)1.55Xf1254araLTranscriptional regulator (ARAC Family)-1.10Xf2344furTranscriptional regulator (FUR Family)1.19Xf2336colRTwo-component system  regulatory protein1.32Xf2534colRTwo-component system  regulatory protein-0.95Xf1752Transcriptional regulator (LYSR Family)1.64Xf1733AF0343Tryptophan repressor binding protein1.13Xf1749opdETranscriptional regulator1.65Xf1730yafCTranscriptional regulator (LYSR Family)1.97Sugar-Nucleotide Biosynthesis, ConversionsXf0260xanAPhosphoglucomutase/ Phosphomannomutase0.92Central Intermediary Metabolism—Pool, Multipourpose ConversionsXf0880yadFCarbonic anhydrase-1.30Xf2255acsAcetyl coenzyme A synthetase-1.37Central Intermediary Metabolism—Amino SugarsXf2355Exo II n-acetyl-beta-glucosaminidase1.44Biosynthesis of Small MoleculesAmino Acids Biosynthesis—Aspartate family, pyruvate familyXf2272metE5-methyltetrahydro pteroyltriglutamate–homocysteine methyltransferase1.44Xf1121metF  OR AQ_14295,10-methylene tetrahydrofolate reductase0.92Xf2223thrCThreonine synthase1.00Xf0863met2Homoserine O-acetyltransferase1.25Amino Acids Biosynthesis—Aromatic Amino Acid FamilyXf0624aroEShikimate 5-dehydrogenase1.64Nucleotides Biosynthesis—Salvage of Nucleosides and NucleotidesXf2150apaHDiadenosine tetraphosphatase1.14Xf2354hptHypoxanthine-guanine phosphoribosyl transferase1.08Nucleotides Biosynthesis –2′ DeoxyribonucleotidesXf0580PH1695Thymidylate kinase-0.93Xf1196nrdA OR TP1008Ribonucleoside-diphosphate reductase alpha chain1.10Nucleotides Biosynthesis—Purine RibonucleotidesXf1503gmk OR spoRGuanylate kinase0.86Nucleotides Biosynthesis—Pyrimidine RibonucleotidesXf1107carB OR pyrACarbamoyl-phosphate synthase large chain-0.99Xf1106carACarbamoyl-phosphate synthase small chain-0.96Cofactors, Prosthetic Groups, Carriers Biosynthesis—Menaquinone, UbiquinoneXf1487ubiEUbiquinone menaquinone transferase-1.64Cofactors, Prosthetic Groups, Carriers Biosynthesis—PantothenateXf0229panB3-Methyl-2-oxobutanoate hydroxy methyltransferase-1.50Cofactors, Prosthetic Groups, Carriers Biosynthesis—ThiaminXf0783thiGThiamine   biosynthesis protein-0.87Cofactors, Prosthetic Groups, Carriers Biosynthesis—RiboflavinXf1748MJ06715-amino-6-(5-phospho ribosylamino) uracil reductase1.05Cofactors, Prosthetic Groups, Carriers Biosynthesis—BiotinXf2477bioDDethiobiotin synthetase1.08Cofactors, Prosthetic Groups, Carriers Biosynthesis—OthersXf1916AF1671Coenzime F390 synthetase1.21Fatty Acid and Phosphatidic Acid BiosynthesisXf2269DRB00803-alpha-hydroxysteroid dehydrogenase-0.93Xf0572fabABeta-hydroxydecanoyl-ACP dehydratase1.18Macromolecule MetabolismDNA metabolism—ReplicationXf0001dnaAChromosomal replication initiator-1.02Xf0002dnaNDNA polymerase III, beta chain-1.39Xf0002dnaNDNA polymerase III, beta chain-1.15Xfa0003topA OR supXTopoisomerase I-1.60Xf1353parCTopoisomerase subunit0.98DNA metabolism—RecombinationXf0425recDExodeoxyribonuclease V alpha chain-0.96Xf0425recDExodeoxyribonuclease V alpha chain-1.02Xf0423ecb OR rorAExodeoxyribonuclease V beta chain1.30DNA metabolism—RepairXf1902ruvB OR HL0312Holliday junction binding protein, DNA helicase-1.20Xf2692ungUracil-DNA glycosylase-1.18DNA Metabolism—Restriction, ModificationXf0935LLAIIAMethyltransferase-0.83Xf1804SPHIMSite-specific DNA-methyltransferase1.12Xf1774hpaIIMDNA methyltransferase-0.81DNA Metabolism—Structural DNA Binding ProteinsXf0446bbh3DNA-binding protein-1.19Xf1644ssbSingle-stranded DNA binding protein1.05RNA Metabolism—Ribosomes—Maturation and ModificationXf0441rimIRibosomal-protein-alanine acetyl transferase1.87Xf0939rluD OR sfhBRidosomal large subunit pseudoeridine synthase D-1.02RNA Metabolism—Ribosomal ProteinsXf1164rplE OR rpl5 OR HI079050S ribosomal protein L5-0.91Xf0238rpsO OR secC30S ribosomal protein S15-1.23Xf1166rpsH OR rps8 OR HI079230S ribosomal protein S8-1.4Xf1169rpsE OR spc30S ribosomal protein S5-1.14RNA Metabolism—RNA Synthesis, Modification, DNA TranscriptionXf1108greATranscriptional elongation factor-1.73Xf0227pcnBPolynucleotide adenyltransferase-1.31Xf2632rpoC OR tabBRNA polymerase beta subunit1.09Xf2606rluCPseudourylate synthase-1.08RNA Metabolism—Aminoacyl tRNA Synthetases, tRNA ModificationXf0428TM0492Tryptophanyl-tRNA synthetase-1.89Xf0445proS OR drpAProlyl-tRNA synthetase-1.08Xf0134valS OR HI1391Valyl-tRNA synthetase-0.96Xf0169tyrS OR HI1610Tyrosyl-tRNA synthetase1.93Xf1314queAS-Adenosylmethionine  tRNA ribosyltransferase-isomerase-1.00Xf0736thrSThreonyl-tRNA synthetase-1.08RNA Metabolism—RNA DegradationXf1505rphRibonuclease PH-0.74Xf1041rnhBRibonuclease HII-1.00Xf2615rnaSA3Ribonuclease1.00Protein Metabolism—Translation and ModificationXf0644mipPeptidyl-propyl cis-trans isomerase-1.11Xf2629fusAElongation factor G-0.90Protein Metabolism—Protein DegradationXf0220pepQProline dipeptidase-1.13Xf0453hflC OR HI0150Integral membrane proteinase1.65Xf2241mucDPeriplasmic protease-0.87Xf1479ptrB OR tlpPeptidase-0.82Xf2330slpDProteinase-0.85Cell StructureMurein Sacculus, PeptidoglycanXf0416vacJLipoprotein precursor-0.78Xf0799ddlB OR ddlD-Alanine-D-alanine ligase B-1.69Xf0276mplUDP-N-acetylmuramate- L-alanine ligase-0.88Surface StructuresXf0487Fimbrillin1.07Xf2539Fimbrial protein-1.02Xf2544pilBPilus biogenesis protein-0.79Chemotaxis and Mobility—Surface Polysaccharides, Lipopolysaccharides and AntigensXf1289kdsA2-dehydro-3-deoxy phosphooctonate  aldolase-0.90Xf1419lpxD OR firA OR omsAAcetyltransferase1.05Xf1646lpxD OR firAUDP-3-O-(R-3-hydroxy myristoyl)-glucosamine N-acyltransferase-0.75Xf1638Dolichyl-phosphate mannose synthase related protein-1.02Xf0879rfbULipopolysaccharide biosynthesis protein-0.74Xf2154opsXSaccharide biosynthesis regulatory protein-1.00Xf0105kdtA OR waaA3-deoxy-D-manno-octulosonic acid trasnferase1.50Membrane Components—Outer Membrane ConstituentsXf1024Outer membrane protein H.8 precursor-1.19Cellular ProcessesTransport—CationsXf1903kup OR trkDPotassium uptake protein1.01Xf1903kup OR trkDPotassium uptake protein1.40Xf0599ybiLTONB-dependent receptor for iron transport1.46Xf0395bfrBacterioferritin-1.22Transport—Amino Acids, AminesXf1937gltPProton glutamate symport protein-1.00Transport—Protein, Peptide SecretionXf2685sppAProtease IV-0.88Xf2261HI0561 560Oligopeptide transporter-1.12Transport—Carbohydrates, Organic Acids, AlchoholsXf0976dctAC4-dicarboxylate transport protein-1.10Cell DivisionXf2281DR0012Chromosomepartitioning protein-1.08OtherXf2251ppaSolute Na+ symporter-1.64Xf1728F451Transport protein1.11Xf1604btuEABC transporter vitamin B12 uptake permease-1.48Xf1409HI1148ABC transporter ATP-binding protein0.84Mobile Genetic ElementsTransposon- and Intron-Related FunctionsXf1775IS629Reverse transcriptase1.06Xf0535Transposase ORFA-0.80Phage-Related Functions and ProphagesXf2522Phage-related protein1.52Xf2522Phage-related protein1.02Xfa0040trbIConjugal transfer protein-0.98Xf2291Phage-related protein0.95Xf0513lycVPhage-related endolysin-1.52Xf1786Phage-related protein1.32Xf1706GP37Phage-related tail fiber protein1.31Xf0685Phage-related protein0.86Xf0704Phage-related protein1.18Xf1875Phage-related protein1.44Plasmid-Related FunctionsXfa0006traA OR virB3Conjugal transfer protein-1.13Xfa0013traAO OR virB9Conjugal transfer protein-1.37Xfa0008traAC ORvirB5Conjugal transfer protein-1.54Pathogenicity,Virulence, and AdaptationToxin production and detoxificationXf0262cvaCColicin V precursor7.29Xf0263cvaCColicin V precursor1.70Xf1011frpCHemolysin-type calcium binding  protein-1.45Xf1827ohrOrganic hydroperoxide resistance protein-1.43Xf2614sodA OR sodSuperoxide dismutase (MN)-1.47Xf1210gst OR HI0111Glutathione S-transferase-1.00Xf1890gpoGlutathione peroxidase-like protein0.86Xf2135frnEPolyketide synthase  (PKS)1.80Xf1897tolBTOLB protein precursor-1.30Xf1729DR1890Phenylacetaldehyde dehydrogenase0.91Host Cell Wall DegradationXf0818engXCAEndo-1,4-beta-glucanase-0.89Adaptation Atypical ConditionXf2682mdoGPeriplasmic glucan biosynthesis protein-0.80Xf2622tapBTemperature acclimation protein B-1.30Surface ProteinsXf1516uspA1Surface-exposed outer membrane protein-1.28ExopolysaccharydesXf2360gumMGumm protein-1.08OtherXf1529hsfSurface protein1.96Xf1532oxyROxidative stress transcriptional regulator0.96Xf2121vapEVirulence-associated protein E1.24Xf1987vacBVACB protein-1.35Xf1114rpfCRegulator of pathogenicity factors-0.87ORFs with Undefined CategoryXf1723yrpGSugar-phosphate dehydrogenase1.30Xf0088hflXGTP-binding protein1.36Hypothetical ProteinsXf1287Hypothetical protein1.40Xf0493Hypothetical protein0.94Xf0037Hypothetical protein-1.11Xf1655Hypothetical protein0.82Xf0726Hypothetical protein-1.17Xf1835Hypothetical protein-0.85Xfa0031Hypothetical protein-1.60Xf2413Hypothetical protein0.96Xf0871Hypothetical protein1.69Xf2454Hypothetical protein-0.97Xf1769Hypothetical protein-0.80Xf1803Hypothetical protein-2.00Xf0512Hypothetical protein-0.93Xf0531Hypothetical protein-1.72Xf1868Hypothetical protein1.11Xf1881Hypothetical protein1.18Xf0917Hypothetical protein1.25Xf1738Hypothetical protein1.37Xf0242Hypothetical protein1.27Xf1228Hypothetical protein1.01Xf1279Hypothetical protein1.11Xf1575Hypothetical protein1.14Xf2597Hypothetical protein-0.94Xf0516Hypothetical protein1.16Xf2017Hypothetical protein-1.51Xf1989Hypothetical protein-0.94Xf2410Hypothetical protein-1.60Xf2304Hypothetical protein-1.26Xf0959Hypothetical protein1.24Xf2115Hypothetical protein1.23Xf1100Hypothetical protein1.04Xf1704Hypothetical protein0.95Xf0974Hypothetical protein-1.26Xf0491Hypothetical protein1.31Xf1060Hypothetical protein1.77Xf2151Hypothetical protein1.73Xf2449Hypothetical protein-1.01Xf2305Hypothetical protein-0.77Xf1721Hypothetical protein1.14Xf0626Hypothetical protein-1.39Xf2411Hypothetical protein1.01Xf1770Hypothetical protein-0.87Xf1364Hypothetical protein-0.85Xf1710Hypothetical protein0.90Xf1761Hypothetical protein1.44Xf1787Hypothetical protein1.38Xf0540Hypothetical protein-1.30Xf1788Hypothetical protein1.06Xf0646Hypothetical protein1.03Xf2543Hypothetical protein-0.98Xf0914Hypothetical protein-1.33Xf2702Hypothetical protein-1.52Xf0492Hypothetical protein1.55Xf1239Hypothetical protein1.01Xf0074Hypothetical protein-1.07Xfa0004Hypothetical protein-1.78Xf1687Hypothetical protein1.32Xf0388Hypothetical protein-0.86Xf0025Hypothetical protein-1.23Xf1434Hypothetical protein-1.24Xf2125Hypothetical protein0.89Xf1513Hypothetical protein1.18Xf2711Hypothetical protein1.23Xf0035Hypothetical protein1.31Xf1441Hypothetical protein-1.41Xf2514Hypothetical protein1.71Xf2626Hypothetical protein1.44Xf0687Hypothetical protein1.07Xf1917Hypothetical protein1.90Xf2271Hypothetical protein1.50Xf1036Hypothetical protein-0.99Xfa0017Hypothetical protein-1.98Xf0529Hypothetical protein1.09Xf2103Hypothetical protein-1.05Xf1986Hypothetical protein-1.05Xf1700Hypothetical protein1.12Xf1719Hypothetical protein1.08Xf1753Hypothetical protein1.44Xf0019Hypothetical protein0.85Xf0293Hypothetical protein-1.15Xf0300Hypothetical protein1.67Xf0279Hypothetical protein1.79Xf0735Hypothetical protein-0.94Xf1010Hypothetical protein-0.97Xf1580Hypothetical protein0.80Xf2021Hypothetical protein1.21Xf2738Hypothetical protein1.49Xf0877Hypothetical protein1.28Xf2270Hypothetical protein1.13Xf0488Hypothetical protein1.50Xf0264Hypothetical protein4.10Xf2701Hypothetical protein-1.68Xf2768Hypothetical protein1.35Xf0688Hypothetical protein0.96Xf0898Hypothetical protein1.15Xf0426Hypothetical protein-1.23Xf0443Hypothetical protein-1.06Xf1421Hypothetical protein-1.40Xf2193Hypothetical protein-2.17Xf2390Hypothetical protein1.24Xf1128Hypothetical protein-1.16Xf2116Hypothetical protein1.52Xf0467Hypothetical protein-1.18Xf1193Hypothetical protein-0.80Xf1032Hypothetical protein-1.33Xf2262Hypothetical protein-1.60Conserved Hypothetical ProteinsXfa0045Conserved hypothetical protein-2.22Xf2450Conserved hypothetical protein-1.22Xf2609Conserved hypothetical protein-0.87Xf1754Conserved hypothetical protein1.83Xf0805Conserved hypothetical protein-0.81Xf2493Conserved hypothetical protein1.13Xf2088Conserved hypothetical protein1.26Xf0196Conserved hypothetical protein-1.95Xf1750Conserved hypothetical protein1.36Xf1745Conserved hypothetical protein1.24Xf2647Conserved hypothetical protein1.13Xf2252Conserved hypothetical protein-2.81Xf2010Conserved hypothetical protein-1.06Xf2237Conserved hypothetical protein-0.85Xfa0032SCJ21.16Conserved hypothetical protein-1.06Xf0758yjeEConserved hypothetical protein-1.40Xf0407yccWConserved hypothetical protein0.98Xf0552yraLConserved hypothetical protein-0.92Xf2651ycbYConserved hypothetical protein-1.19Xf2575DR0386Conserved hypothetical protein-0.86Xf0363yiaDConserved hypothetical protein-1.78Xf0066ylbKConserved hypothetical protein1.10Xf2179ybeNConserved hypothetical protein1.29Xf2153HI0260.1Conserved hypothetical protein-1.14Xf0553HI1655Conserved hypothetical protein-1.29Xf2014DR0566Conserved hypothetical protein1.14Xf0139yjgPConserved hypothetical protein1.15Xf2474yjeKConserved hypothetical protein-0.79Xf2096MTH1196Conserved hypothetical protein-1.93Xf1054TM1087Conserved hypothetical protein-0.91Xf0554yraNConserved hypothetical protein-0.85Xf0339btuB OR bfe OR cerConserved hypothetical protein-0.91Xf1272RV1827 OR MTCY1A11.16CConserved hypothetical protein-1.02Xf1405yhbJConserved hypothetical protein-0.88Xf1808ybaBConserved hypothetical protein-1.19Xf1829RP471Conserved hypothetical protein-1.05Xf0941yuxKConserved hypothetical protein-0.80Figure 2
Evaluation of transcriptional modulation of selected genes by Real-Time qPCR. In order to confirm the reliability of the microarray experiments, 16 genes have been randomly selected and their transcription modulation was verified by Real-Time qPCR. The same RNA samples used in the microarray hybridizations were converted to cDNA and the relative expression ratios (RQ) of these genes have been measured with the aid of specific Taq-Man probes. ORFXf1311, which encodes a rod-shaped determining protein (MreD), has been used as an endogenous control for experimental normalization, since the microarray hybridization experiments showed that this ORF is constitutively expressed in both PW and 3G10R. Variations in transcriptional modulation were calculated having the expression levels in PW as a reference and are represented by the log2 ratio of the relative quantifications (RQ). Experiments were performed in triplicate and graphic shows the average values and their respective standard deviations.Interestingly, we were able to verify that several genes directly associated with pathogenicity, virulence and adaptation had their transcription modulated in response to growth in xylem-based chemical conditions. This group includes genes associated with adaptation to atypical conditions (such as the temperature acclimatation protein TAPB (ORFXf2622) and the oxidative stress transcriptional regulator OxyR (ORF Xf1532)); surface proteins (including adhesion factors, such as the outer membrane protein Hsf (ORF Xf1529)), and genes involved in toxin production and/or detoxification (such as the colicin precursors encoded by ORFs Xf0262 and Xf0263), among others (see Table 1 for details).The lack of aminoacids in 3G10R also seems to lead to overexpression of at least four genes directly involved in the biosynthesis of such molecules (represented by ORFsXf0624, Xf0863, Xf1121, Xf2223 andXf2272). On the other hand, cells that are grown on the peptide-based diet provided by PW display an increased production of proteolytic enzymes, such as MucD (ORF Xf2241), PtrB (ORF Xf1479) and PepQ peptidase (ORF Xf0220), which has been shown to play a major role in lactic acid bacteria, providing the cells with amino acids derived from extracellular protein sources during milk fermentation [28].The transcriptome results also show that the elevated growth rate ofX. fastidiosa cells kept in PW is associated with the upregulation of several genes involved in a series of metabolic pathways and processes that are important to sustain continued bacterial growth [29]. These include ORFs associated with DNA replication, recombination and repair, such as dnaA (the chromosomal replication initiator, encoded by ORF Xf0001), dnaN (the β chain of DNA polymerase III, encoded by ORF Xf0002), recD (the alpha chain of exodeoxyribonuclease V, encoded by ORF Xf0425), ruvB (a Holiday junction-associated helicase, encoded by ORF Xf1902) and ung (an uracil-DNA glycosilase, encoded by ORF Xf2692).However, since elevated growth rates establish a higher demand for energy consumption, they can only be maintained if ATP production is increased. Thus, it is interesting to verify that growth in PW is associated with overexpression of several genes involved in all major steps of the central metabolic pathway, such as triose phosphate isomerase (Xf0303) (glycolytic pathway); succinyl-coA synthase (Xf2548) and fumarate hydratase C (Xf1554) (Krebs cycle), as well as genes from the nuo operon (represented by ORFs Xf0308, Xf0310, Xf0311 and Xf0317, resp.). Genes from this operon encode subunits of the NADH Dehydrogenase I complex, the first component of the respiratory electron transport chain. Interestingly, coordinated overexpression of such genes has already been shown to occur in E. coli cells submitted to differing culture conditions [30, 31].


### 3.2. Increased Growth Rate in PW Is Associated with Upregulation of Genes from the Electron Transport Chain and Consequent Enhancement of Respiratory Activity

As mentioned before, PW is the most commonly used medium to cultivateXylella fastidiosa under laboratory conditions, since this formulation has been shown to sustain efficient growth of all isolates of this phytobacterium [11]. Thus, the positive modulation of genes directly involved in oxidative phosphorylation, might lead to increased aerobic respiratory activity and consequent ATP production, which seems to greatly improve on the fastidious nature of this bacterium. Thus, we decided to verify O2 consumption in PW-grown cells as a way to indirectly estimate the activation of aerobic respiration in X. fastidiosa. This experiment allowed us to verify not only the activation of the aerobic respiration, but also to obtain biological confirmation of a major metabolic change originally predicted solely on the transcriptome data.As shown in Figure3, X. fastidiosa cells transferred from PW to 3G10R displayed a continued decrease in the respiratory rate, which is unaffected in cells transferred to fresh PW medium. A direct comparison between the results observed for the PW culture, at day 3, and the 3G10R culture, at day 13, (the same time points used for transcriptome comparisons) shows that cells grown in PW display overexpression of several genes involved in all major steps of the central metabolic pathway, as well as a respiratory rate that is about five times greater than that observed with cells grown in 3G10R. Thus, the results from this experiment confirmed that there is a significant increase in oxidative phosphorylation when X. fastidiosa cells are grown in PW (as previously inferred from the analysis of transcriptome data), which helps to explain the effectiveness of this culture medium in sustaining continued and vigorous growth of X. fastidiosa strains.Figure 3
Evaluation of respiratory rates inXylella fastidiosa cells growing in PW and 3G10R. X. fastidiosa cells were grown into middle exponential phase in PW and subsequently transferred (in a 1 : 20 proportion) into fresh PW and 3G10R cultures. Bacterial growth in both cultures was monitored through OD600 measurements and aliquots were taken from each culture to evaluate O2 consumption with the aid of an oxygraph in intact cells. Respiratory rate for each culture was calculated as the ratio between O2 consumption and the respective OD600 value obtained at each time point. Measurements were taken until day 7 (in PW) and day 13 (in 3G10R). Experiments were performed in triplicate and graphic shows the average values and their respective standard deviations.


## 3.1. X. fastidiosa Cells Growing in PW and 3G10R Display Distinct Growth Patterns and Different Transcriptome Profiles

To evaluate the behavior ofX. fastidiosa cells under xylem-based chemistry conditions, bacterial cultures were monitored in both 3G10R and PW, the complex medium traditionally used to cultivate this bacterium in the laboratory. As observed in Figure 1, PW cultures reached higher cellular densities (OD600~0.3) in a shorter period of time (4 days) when compared to cells grown in 3G10R, which had to be cultivated for a period of 14 days in order to reach a similar cellular density (OD600~0.25). Moreover, although 3G10R cultures exhibited continuous growth over the course of the experiment, they failed to display the typical profile of a bacterial growth curve, as observed in PW cultures. Such lack of an exponential growth phase in 3G10R cultures is typically observed in bacteria growing in nutrient-restricted environments, a situation that is likely to resemble xylem conditions [22–26]. Recently, Zaini et al. [27] showed that X. fastidiosa cells grown in pure xylem sap rapidly reach stationary phase without a detectable exponential growth, probably due to nutrient limitation.Figure 1
Xylella  fastidiosa growth patterns in PW and 3G10R media. Both cultures have been made with a 1 : 20 ml inoculum of X. fastidiosa 9a5c cells grown into late exponential phase in PW (OD600 = 0.25). Cultures were then incubated in an orbital shaker at 28°C and 100 rpm. One milliliter (1 ml) aliquots were taken from each culture, on a daily basis, to monitor bacterial growth through OD600 readings. Measurements were performed in triplicate and graphic shows the average values and their respective standard deviations.To evaluate and compare the bacterial transcriptome profiles in PW and 3G10R, samples from the resulting RNAs were used in competitive hybridizations againstX. fastidiosa microarrays, as described by Nunes et al. [19]. Replicated experiments were performed with two independent RNA preparations from cells cultivated in each medium, which resulted in a series of 8 independent readings for each probe spotted in the microarrays, as described in the materials and methods. Statistical analysis of such results revealed a total of 132 genes that displayed overexpression in cells grown in 3G10R, while 167 genes were upregulated in cells grown in PW. These genes, as well as their respective changes in expression ratio are shown in Table 1. More detailed information about these genes can be obtained through the Gene expression Omnibus (GEO) webpage, through Series number GSE 6619 (see http://ncbi.nlm.nih.gov/geo). In order to access the overall reliability of these data, we have confirmed gene expression variation of several genes using an alternative approach. Thus, we performed Real-Time qPCR experiments with the same RNA samples used in the microarray hybridizations, aiming at double-checking the changes in expression of 16 genes present in Table 1 (~5% of all modulated genes). These genes have been randomly chosen from different functional categories and all displayed average expression ratios that correlate with the microarray results (see Figure 2).Table 1
List of genes that displayed statistically significant variation in gene expression. Genes with positive Log2 ratio are overexpressed in 3G10R, while genes with negative Log2 ratio are overexpressed in PW.
Functional GroupORFGeneGene ProductLog2NumberName(3G10R/PW)Intermediary MetabolismEnergy metabolism, carbon—Aerobic respirationXf0308nuoDNADH-ubiquinone oxidoreductase, NQO4 subunit-0.93Xf0310nuoFNADH-ubiquinone oxidoreductase, NQO1 subunit-1.08Xf0311nuoGNADH-ubiquinone oxidoreductase, NQO3 subunit-1.14Xf0317nuoMNADH-ubiquinone oxidoreductase, NQO13 subunit-1.03Xf0347dld1D-Lactate dehydrogenase1.18Energy metabolism, carbon—GlycolysisXf0303tpiA OR tpiTriosephosphate isomerase-0.89Energy metabolism, carbon—TCA cycleXf2548sucDSuccinyl-CoA synthetase, alpha subunit-1.67Xf1554fumCFumarate hydratase-1.47Xf1554fumCFumarate hydratase-1.36Energy metabolism, carbon—Electron TransportXf1990yneNThioredoxin-1.14Xf0620dsbDc-Type cytochrome biogenesis protein (Copper Tolerance)-0.83Degradation—Degradation of Small MoleculesXf1250rocFArginine deaminase-2.00Xf1740yliIGlucose dehydrogenase B1.45Xf2395axeAAcetylxylan esterase1.75Xf2432gtaBUTP-glucose-1-phosphate uridylyl transferase-1.14Xf0610galEUDP-glucose 4-epimerase-1.44Xf2210Dioxygenase1.00Regulatory FunctionsXf1354yybATranscriptional regulator (MARR Family)1.27Xf1354yybATranscriptional regulator (MARR Family)1.55Xf1254araLTranscriptional regulator (ARAC Family)-1.10Xf2344furTranscriptional regulator (FUR Family)1.19Xf2336colRTwo-component system  regulatory protein1.32Xf2534colRTwo-component system  regulatory protein-0.95Xf1752Transcriptional regulator (LYSR Family)1.64Xf1733AF0343Tryptophan repressor binding protein1.13Xf1749opdETranscriptional regulator1.65Xf1730yafCTranscriptional regulator (LYSR Family)1.97Sugar-Nucleotide Biosynthesis, ConversionsXf0260xanAPhosphoglucomutase/ Phosphomannomutase0.92Central Intermediary Metabolism—Pool, Multipourpose ConversionsXf0880yadFCarbonic anhydrase-1.30Xf2255acsAcetyl coenzyme A synthetase-1.37Central Intermediary Metabolism—Amino SugarsXf2355Exo II n-acetyl-beta-glucosaminidase1.44Biosynthesis of Small MoleculesAmino Acids Biosynthesis—Aspartate family, pyruvate familyXf2272metE5-methyltetrahydro pteroyltriglutamate–homocysteine methyltransferase1.44Xf1121metF  OR AQ_14295,10-methylene tetrahydrofolate reductase0.92Xf2223thrCThreonine synthase1.00Xf0863met2Homoserine O-acetyltransferase1.25Amino Acids Biosynthesis—Aromatic Amino Acid FamilyXf0624aroEShikimate 5-dehydrogenase1.64Nucleotides Biosynthesis—Salvage of Nucleosides and NucleotidesXf2150apaHDiadenosine tetraphosphatase1.14Xf2354hptHypoxanthine-guanine phosphoribosyl transferase1.08Nucleotides Biosynthesis –2′ DeoxyribonucleotidesXf0580PH1695Thymidylate kinase-0.93Xf1196nrdA OR TP1008Ribonucleoside-diphosphate reductase alpha chain1.10Nucleotides Biosynthesis—Purine RibonucleotidesXf1503gmk OR spoRGuanylate kinase0.86Nucleotides Biosynthesis—Pyrimidine RibonucleotidesXf1107carB OR pyrACarbamoyl-phosphate synthase large chain-0.99Xf1106carACarbamoyl-phosphate synthase small chain-0.96Cofactors, Prosthetic Groups, Carriers Biosynthesis—Menaquinone, UbiquinoneXf1487ubiEUbiquinone menaquinone transferase-1.64Cofactors, Prosthetic Groups, Carriers Biosynthesis—PantothenateXf0229panB3-Methyl-2-oxobutanoate hydroxy methyltransferase-1.50Cofactors, Prosthetic Groups, Carriers Biosynthesis—ThiaminXf0783thiGThiamine   biosynthesis protein-0.87Cofactors, Prosthetic Groups, Carriers Biosynthesis—RiboflavinXf1748MJ06715-amino-6-(5-phospho ribosylamino) uracil reductase1.05Cofactors, Prosthetic Groups, Carriers Biosynthesis—BiotinXf2477bioDDethiobiotin synthetase1.08Cofactors, Prosthetic Groups, Carriers Biosynthesis—OthersXf1916AF1671Coenzime F390 synthetase1.21Fatty Acid and Phosphatidic Acid BiosynthesisXf2269DRB00803-alpha-hydroxysteroid dehydrogenase-0.93Xf0572fabABeta-hydroxydecanoyl-ACP dehydratase1.18Macromolecule MetabolismDNA metabolism—ReplicationXf0001dnaAChromosomal replication initiator-1.02Xf0002dnaNDNA polymerase III, beta chain-1.39Xf0002dnaNDNA polymerase III, beta chain-1.15Xfa0003topA OR supXTopoisomerase I-1.60Xf1353parCTopoisomerase subunit0.98DNA metabolism—RecombinationXf0425recDExodeoxyribonuclease V alpha chain-0.96Xf0425recDExodeoxyribonuclease V alpha chain-1.02Xf0423ecb OR rorAExodeoxyribonuclease V beta chain1.30DNA metabolism—RepairXf1902ruvB OR HL0312Holliday junction binding protein, DNA helicase-1.20Xf2692ungUracil-DNA glycosylase-1.18DNA Metabolism—Restriction, ModificationXf0935LLAIIAMethyltransferase-0.83Xf1804SPHIMSite-specific DNA-methyltransferase1.12Xf1774hpaIIMDNA methyltransferase-0.81DNA Metabolism—Structural DNA Binding ProteinsXf0446bbh3DNA-binding protein-1.19Xf1644ssbSingle-stranded DNA binding protein1.05RNA Metabolism—Ribosomes—Maturation and ModificationXf0441rimIRibosomal-protein-alanine acetyl transferase1.87Xf0939rluD OR sfhBRidosomal large subunit pseudoeridine synthase D-1.02RNA Metabolism—Ribosomal ProteinsXf1164rplE OR rpl5 OR HI079050S ribosomal protein L5-0.91Xf0238rpsO OR secC30S ribosomal protein S15-1.23Xf1166rpsH OR rps8 OR HI079230S ribosomal protein S8-1.4Xf1169rpsE OR spc30S ribosomal protein S5-1.14RNA Metabolism—RNA Synthesis, Modification, DNA TranscriptionXf1108greATranscriptional elongation factor-1.73Xf0227pcnBPolynucleotide adenyltransferase-1.31Xf2632rpoC OR tabBRNA polymerase beta subunit1.09Xf2606rluCPseudourylate synthase-1.08RNA Metabolism—Aminoacyl tRNA Synthetases, tRNA ModificationXf0428TM0492Tryptophanyl-tRNA synthetase-1.89Xf0445proS OR drpAProlyl-tRNA synthetase-1.08Xf0134valS OR HI1391Valyl-tRNA synthetase-0.96Xf0169tyrS OR HI1610Tyrosyl-tRNA synthetase1.93Xf1314queAS-Adenosylmethionine  tRNA ribosyltransferase-isomerase-1.00Xf0736thrSThreonyl-tRNA synthetase-1.08RNA Metabolism—RNA DegradationXf1505rphRibonuclease PH-0.74Xf1041rnhBRibonuclease HII-1.00Xf2615rnaSA3Ribonuclease1.00Protein Metabolism—Translation and ModificationXf0644mipPeptidyl-propyl cis-trans isomerase-1.11Xf2629fusAElongation factor G-0.90Protein Metabolism—Protein DegradationXf0220pepQProline dipeptidase-1.13Xf0453hflC OR HI0150Integral membrane proteinase1.65Xf2241mucDPeriplasmic protease-0.87Xf1479ptrB OR tlpPeptidase-0.82Xf2330slpDProteinase-0.85Cell StructureMurein Sacculus, PeptidoglycanXf0416vacJLipoprotein precursor-0.78Xf0799ddlB OR ddlD-Alanine-D-alanine ligase B-1.69Xf0276mplUDP-N-acetylmuramate- L-alanine ligase-0.88Surface StructuresXf0487Fimbrillin1.07Xf2539Fimbrial protein-1.02Xf2544pilBPilus biogenesis protein-0.79Chemotaxis and Mobility—Surface Polysaccharides, Lipopolysaccharides and AntigensXf1289kdsA2-dehydro-3-deoxy phosphooctonate  aldolase-0.90Xf1419lpxD OR firA OR omsAAcetyltransferase1.05Xf1646lpxD OR firAUDP-3-O-(R-3-hydroxy myristoyl)-glucosamine N-acyltransferase-0.75Xf1638Dolichyl-phosphate mannose synthase related protein-1.02Xf0879rfbULipopolysaccharide biosynthesis protein-0.74Xf2154opsXSaccharide biosynthesis regulatory protein-1.00Xf0105kdtA OR waaA3-deoxy-D-manno-octulosonic acid trasnferase1.50Membrane Components—Outer Membrane ConstituentsXf1024Outer membrane protein H.8 precursor-1.19Cellular ProcessesTransport—CationsXf1903kup OR trkDPotassium uptake protein1.01Xf1903kup OR trkDPotassium uptake protein1.40Xf0599ybiLTONB-dependent receptor for iron transport1.46Xf0395bfrBacterioferritin-1.22Transport—Amino Acids, AminesXf1937gltPProton glutamate symport protein-1.00Transport—Protein, Peptide SecretionXf2685sppAProtease IV-0.88Xf2261HI0561 560Oligopeptide transporter-1.12Transport—Carbohydrates, Organic Acids, AlchoholsXf0976dctAC4-dicarboxylate transport protein-1.10Cell DivisionXf2281DR0012Chromosomepartitioning protein-1.08OtherXf2251ppaSolute Na+ symporter-1.64Xf1728F451Transport protein1.11Xf1604btuEABC transporter vitamin B12 uptake permease-1.48Xf1409HI1148ABC transporter ATP-binding protein0.84Mobile Genetic ElementsTransposon- and Intron-Related FunctionsXf1775IS629Reverse transcriptase1.06Xf0535Transposase ORFA-0.80Phage-Related Functions and ProphagesXf2522Phage-related protein1.52Xf2522Phage-related protein1.02Xfa0040trbIConjugal transfer protein-0.98Xf2291Phage-related protein0.95Xf0513lycVPhage-related endolysin-1.52Xf1786Phage-related protein1.32Xf1706GP37Phage-related tail fiber protein1.31Xf0685Phage-related protein0.86Xf0704Phage-related protein1.18Xf1875Phage-related protein1.44Plasmid-Related FunctionsXfa0006traA OR virB3Conjugal transfer protein-1.13Xfa0013traAO OR virB9Conjugal transfer protein-1.37Xfa0008traAC ORvirB5Conjugal transfer protein-1.54Pathogenicity,Virulence, and AdaptationToxin production and detoxificationXf0262cvaCColicin V precursor7.29Xf0263cvaCColicin V precursor1.70Xf1011frpCHemolysin-type calcium binding  protein-1.45Xf1827ohrOrganic hydroperoxide resistance protein-1.43Xf2614sodA OR sodSuperoxide dismutase (MN)-1.47Xf1210gst OR HI0111Glutathione S-transferase-1.00Xf1890gpoGlutathione peroxidase-like protein0.86Xf2135frnEPolyketide synthase  (PKS)1.80Xf1897tolBTOLB protein precursor-1.30Xf1729DR1890Phenylacetaldehyde dehydrogenase0.91Host Cell Wall DegradationXf0818engXCAEndo-1,4-beta-glucanase-0.89Adaptation Atypical ConditionXf2682mdoGPeriplasmic glucan biosynthesis protein-0.80Xf2622tapBTemperature acclimation protein B-1.30Surface ProteinsXf1516uspA1Surface-exposed outer membrane protein-1.28ExopolysaccharydesXf2360gumMGumm protein-1.08OtherXf1529hsfSurface protein1.96Xf1532oxyROxidative stress transcriptional regulator0.96Xf2121vapEVirulence-associated protein E1.24Xf1987vacBVACB protein-1.35Xf1114rpfCRegulator of pathogenicity factors-0.87ORFs with Undefined CategoryXf1723yrpGSugar-phosphate dehydrogenase1.30Xf0088hflXGTP-binding protein1.36Hypothetical ProteinsXf1287Hypothetical protein1.40Xf0493Hypothetical protein0.94Xf0037Hypothetical protein-1.11Xf1655Hypothetical protein0.82Xf0726Hypothetical protein-1.17Xf1835Hypothetical protein-0.85Xfa0031Hypothetical protein-1.60Xf2413Hypothetical protein0.96Xf0871Hypothetical protein1.69Xf2454Hypothetical protein-0.97Xf1769Hypothetical protein-0.80Xf1803Hypothetical protein-2.00Xf0512Hypothetical protein-0.93Xf0531Hypothetical protein-1.72Xf1868Hypothetical protein1.11Xf1881Hypothetical protein1.18Xf0917Hypothetical protein1.25Xf1738Hypothetical protein1.37Xf0242Hypothetical protein1.27Xf1228Hypothetical protein1.01Xf1279Hypothetical protein1.11Xf1575Hypothetical protein1.14Xf2597Hypothetical protein-0.94Xf0516Hypothetical protein1.16Xf2017Hypothetical protein-1.51Xf1989Hypothetical protein-0.94Xf2410Hypothetical protein-1.60Xf2304Hypothetical protein-1.26Xf0959Hypothetical protein1.24Xf2115Hypothetical protein1.23Xf1100Hypothetical protein1.04Xf1704Hypothetical protein0.95Xf0974Hypothetical protein-1.26Xf0491Hypothetical protein1.31Xf1060Hypothetical protein1.77Xf2151Hypothetical protein1.73Xf2449Hypothetical protein-1.01Xf2305Hypothetical protein-0.77Xf1721Hypothetical protein1.14Xf0626Hypothetical protein-1.39Xf2411Hypothetical protein1.01Xf1770Hypothetical protein-0.87Xf1364Hypothetical protein-0.85Xf1710Hypothetical protein0.90Xf1761Hypothetical protein1.44Xf1787Hypothetical protein1.38Xf0540Hypothetical protein-1.30Xf1788Hypothetical protein1.06Xf0646Hypothetical protein1.03Xf2543Hypothetical protein-0.98Xf0914Hypothetical protein-1.33Xf2702Hypothetical protein-1.52Xf0492Hypothetical protein1.55Xf1239Hypothetical protein1.01Xf0074Hypothetical protein-1.07Xfa0004Hypothetical protein-1.78Xf1687Hypothetical protein1.32Xf0388Hypothetical protein-0.86Xf0025Hypothetical protein-1.23Xf1434Hypothetical protein-1.24Xf2125Hypothetical protein0.89Xf1513Hypothetical protein1.18Xf2711Hypothetical protein1.23Xf0035Hypothetical protein1.31Xf1441Hypothetical protein-1.41Xf2514Hypothetical protein1.71Xf2626Hypothetical protein1.44Xf0687Hypothetical protein1.07Xf1917Hypothetical protein1.90Xf2271Hypothetical protein1.50Xf1036Hypothetical protein-0.99Xfa0017Hypothetical protein-1.98Xf0529Hypothetical protein1.09Xf2103Hypothetical protein-1.05Xf1986Hypothetical protein-1.05Xf1700Hypothetical protein1.12Xf1719Hypothetical protein1.08Xf1753Hypothetical protein1.44Xf0019Hypothetical protein0.85Xf0293Hypothetical protein-1.15Xf0300Hypothetical protein1.67Xf0279Hypothetical protein1.79Xf0735Hypothetical protein-0.94Xf1010Hypothetical protein-0.97Xf1580Hypothetical protein0.80Xf2021Hypothetical protein1.21Xf2738Hypothetical protein1.49Xf0877Hypothetical protein1.28Xf2270Hypothetical protein1.13Xf0488Hypothetical protein1.50Xf0264Hypothetical protein4.10Xf2701Hypothetical protein-1.68Xf2768Hypothetical protein1.35Xf0688Hypothetical protein0.96Xf0898Hypothetical protein1.15Xf0426Hypothetical protein-1.23Xf0443Hypothetical protein-1.06Xf1421Hypothetical protein-1.40Xf2193Hypothetical protein-2.17Xf2390Hypothetical protein1.24Xf1128Hypothetical protein-1.16Xf2116Hypothetical protein1.52Xf0467Hypothetical protein-1.18Xf1193Hypothetical protein-0.80Xf1032Hypothetical protein-1.33Xf2262Hypothetical protein-1.60Conserved Hypothetical ProteinsXfa0045Conserved hypothetical protein-2.22Xf2450Conserved hypothetical protein-1.22Xf2609Conserved hypothetical protein-0.87Xf1754Conserved hypothetical protein1.83Xf0805Conserved hypothetical protein-0.81Xf2493Conserved hypothetical protein1.13Xf2088Conserved hypothetical protein1.26Xf0196Conserved hypothetical protein-1.95Xf1750Conserved hypothetical protein1.36Xf1745Conserved hypothetical protein1.24Xf2647Conserved hypothetical protein1.13Xf2252Conserved hypothetical protein-2.81Xf2010Conserved hypothetical protein-1.06Xf2237Conserved hypothetical protein-0.85Xfa0032SCJ21.16Conserved hypothetical protein-1.06Xf0758yjeEConserved hypothetical protein-1.40Xf0407yccWConserved hypothetical protein0.98Xf0552yraLConserved hypothetical protein-0.92Xf2651ycbYConserved hypothetical protein-1.19Xf2575DR0386Conserved hypothetical protein-0.86Xf0363yiaDConserved hypothetical protein-1.78Xf0066ylbKConserved hypothetical protein1.10Xf2179ybeNConserved hypothetical protein1.29Xf2153HI0260.1Conserved hypothetical protein-1.14Xf0553HI1655Conserved hypothetical protein-1.29Xf2014DR0566Conserved hypothetical protein1.14Xf0139yjgPConserved hypothetical protein1.15Xf2474yjeKConserved hypothetical protein-0.79Xf2096MTH1196Conserved hypothetical protein-1.93Xf1054TM1087Conserved hypothetical protein-0.91Xf0554yraNConserved hypothetical protein-0.85Xf0339btuB OR bfe OR cerConserved hypothetical protein-0.91Xf1272RV1827 OR MTCY1A11.16CConserved hypothetical protein-1.02Xf1405yhbJConserved hypothetical protein-0.88Xf1808ybaBConserved hypothetical protein-1.19Xf1829RP471Conserved hypothetical protein-1.05Xf0941yuxKConserved hypothetical protein-0.80Figure 2
Evaluation of transcriptional modulation of selected genes by Real-Time qPCR. In order to confirm the reliability of the microarray experiments, 16 genes have been randomly selected and their transcription modulation was verified by Real-Time qPCR. The same RNA samples used in the microarray hybridizations were converted to cDNA and the relative expression ratios (RQ) of these genes have been measured with the aid of specific Taq-Man probes. ORFXf1311, which encodes a rod-shaped determining protein (MreD), has been used as an endogenous control for experimental normalization, since the microarray hybridization experiments showed that this ORF is constitutively expressed in both PW and 3G10R. Variations in transcriptional modulation were calculated having the expression levels in PW as a reference and are represented by the log2 ratio of the relative quantifications (RQ). Experiments were performed in triplicate and graphic shows the average values and their respective standard deviations.Interestingly, we were able to verify that several genes directly associated with pathogenicity, virulence and adaptation had their transcription modulated in response to growth in xylem-based chemical conditions. This group includes genes associated with adaptation to atypical conditions (such as the temperature acclimatation protein TAPB (ORFXf2622) and the oxidative stress transcriptional regulator OxyR (ORF Xf1532)); surface proteins (including adhesion factors, such as the outer membrane protein Hsf (ORF Xf1529)), and genes involved in toxin production and/or detoxification (such as the colicin precursors encoded by ORFs Xf0262 and Xf0263), among others (see Table 1 for details).The lack of aminoacids in 3G10R also seems to lead to overexpression of at least four genes directly involved in the biosynthesis of such molecules (represented by ORFsXf0624, Xf0863, Xf1121, Xf2223 andXf2272). On the other hand, cells that are grown on the peptide-based diet provided by PW display an increased production of proteolytic enzymes, such as MucD (ORF Xf2241), PtrB (ORF Xf1479) and PepQ peptidase (ORF Xf0220), which has been shown to play a major role in lactic acid bacteria, providing the cells with amino acids derived from extracellular protein sources during milk fermentation [28].The transcriptome results also show that the elevated growth rate ofX. fastidiosa cells kept in PW is associated with the upregulation of several genes involved in a series of metabolic pathways and processes that are important to sustain continued bacterial growth [29]. These include ORFs associated with DNA replication, recombination and repair, such as dnaA (the chromosomal replication initiator, encoded by ORF Xf0001), dnaN (the β chain of DNA polymerase III, encoded by ORF Xf0002), recD (the alpha chain of exodeoxyribonuclease V, encoded by ORF Xf0425), ruvB (a Holiday junction-associated helicase, encoded by ORF Xf1902) and ung (an uracil-DNA glycosilase, encoded by ORF Xf2692).However, since elevated growth rates establish a higher demand for energy consumption, they can only be maintained if ATP production is increased. Thus, it is interesting to verify that growth in PW is associated with overexpression of several genes involved in all major steps of the central metabolic pathway, such as triose phosphate isomerase (Xf0303) (glycolytic pathway); succinyl-coA synthase (Xf2548) and fumarate hydratase C (Xf1554) (Krebs cycle), as well as genes from the nuo operon (represented by ORFs Xf0308, Xf0310, Xf0311 and Xf0317, resp.). Genes from this operon encode subunits of the NADH Dehydrogenase I complex, the first component of the respiratory electron transport chain. Interestingly, coordinated overexpression of such genes has already been shown to occur in E. coli cells submitted to differing culture conditions [30, 31].


## 3.2. Increased Growth Rate in PW Is Associated with Upregulation of Genes from the Electron Transport Chain and Consequent Enhancement of Respiratory Activity

As mentioned before, PW is the most commonly used medium to cultivateXylella fastidiosa under laboratory conditions, since this formulation has been shown to sustain efficient growth of all isolates of this phytobacterium [11]. Thus, the positive modulation of genes directly involved in oxidative phosphorylation, might lead to increased aerobic respiratory activity and consequent ATP production, which seems to greatly improve on the fastidious nature of this bacterium. Thus, we decided to verify O2 consumption in PW-grown cells as a way to indirectly estimate the activation of aerobic respiration in X. fastidiosa. This experiment allowed us to verify not only the activation of the aerobic respiration, but also to obtain biological confirmation of a major metabolic change originally predicted solely on the transcriptome data.As shown in Figure3, X. fastidiosa cells transferred from PW to 3G10R displayed a continued decrease in the respiratory rate, which is unaffected in cells transferred to fresh PW medium. A direct comparison between the results observed for the PW culture, at day 3, and the 3G10R culture, at day 13, (the same time points used for transcriptome comparisons) shows that cells grown in PW display overexpression of several genes involved in all major steps of the central metabolic pathway, as well as a respiratory rate that is about five times greater than that observed with cells grown in 3G10R. Thus, the results from this experiment confirmed that there is a significant increase in oxidative phosphorylation when X. fastidiosa cells are grown in PW (as previously inferred from the analysis of transcriptome data), which helps to explain the effectiveness of this culture medium in sustaining continued and vigorous growth of X. fastidiosa strains.Figure 3
Evaluation of respiratory rates inXylella fastidiosa cells growing in PW and 3G10R. X. fastidiosa cells were grown into middle exponential phase in PW and subsequently transferred (in a 1 : 20 proportion) into fresh PW and 3G10R cultures. Bacterial growth in both cultures was monitored through OD600 measurements and aliquots were taken from each culture to evaluate O2 consumption with the aid of an oxygraph in intact cells. Respiratory rate for each culture was calculated as the ratio between O2 consumption and the respective OD600 value obtained at each time point. Measurements were taken until day 7 (in PW) and day 13 (in 3G10R). Experiments were performed in triplicate and graphic shows the average values and their respective standard deviations.


## 4. Discussion

The recent development of xylem-based chemistry media, such as 3G10R, has provided an interesting instrument to study several aspects ofX. fastidiosa behavior under laboratory conditions, where this phytopathogen is typically grown in complex media, such as PW [11]. Interestingly, both PW and 3G10R are capable of sustaining growth of X. fastidiosa cells in vitro, although significant differences have been observed in the bacterial growth rates.Nonetheless, when growing in PW, whereX. fastidiosa cells have been shown to display an increased respiratory rate, as well as an enhanced growth profile, we can observe coordinated upregulation of enzymes from the central metabolic pathway, particularly of the NADH Dehydrogenase I complex, a phenomenon also observed to occur in E. coli grown in different media [30, 31]. This results in strong activation of the aerobic respiratory metabolism, providing the cells with the necessary energy for increased bacterial replication. However, at this point, we do not know the exact mechanism(s) that might be responsible to trigger such a respiratory activation, nor if it plays any role during plant colonization or onset of disease, when the endophytic population of X. fastidiosa seems to increase dramatically inside xylem vessels [32, 33]. It seems unlikely, however, that this metabolic switch occurs only on the account of oxygen concentration, since both cultures were kept under the same aeration conditions during all experimental steps described throughout this work.Incidentally, this situation seems to resemble the fermentative-to-respiratory shift observed inLactococus lactis, a gram-positive, microaerophilic bacterium, with a fermentative metabolism that produces mainly L-lactate from carbohydrates [34, 35]. L. lactis, as well as other members of the Streptococcaceaefamily, such as Streptococcus agalactiae and Enterococcus fecalis, multiply mainly via a fermentative metabolism, even in the presence of oxygen. Curiously, in spite of the fact that these bacteria carry all genes and enzymes necessary to undergo aerobic respiration, prolonged aeration of L. lactis cultures can lead to growth inhibition, DNA degradation and cell death, probably due to the formation of hydrogen peroxide and hydroxyl radicals during aerobic respiration, associated with an incomplete set of oxidative stress-resistance enzymes [36]. However, if exogenous haem is provided during aerated growth, L. lactis cells can undergo a metabolic shunt towards respiratory metabolism, leading to increased ATP production, improved growth and a dramatic increase in long-term survival, when compared to growth in standard fermentation conditions [35]. Further details regarding the fermentation-respiration shift in L. lactis are not completely understood, but it has been documented that the process depends on cytochrome BD (encoded by the cyaBD genes) and is controled by the Catabolite Control Protein (CcpA) [37]. Although more direct evidence is still needed to further clarify this issue, it is tempting to speculate if the presence of hemin chloride in PW might be acting as an exogenous source of haem and activating an analogous mechanism in X. fastidiosa cells that would lead to an increase in aerobic respiration.The observed modulation of triose phosphate isomerase (Xf0303) is also noteworthy, since preliminary studies failed to detect specific activity of several genes from the Glycolytic pathway in bacterial crude extracts, such as aldolase, glyceraldehyde 3-phosphate dehydrogenase and enolase [38]. On the other hand, the activity of glucose 6-phosphate dehydrogenase was detected in these same extracts, leading the authors to suggest that X. fastidiosa cells do not use the glycolytic pathway to oxidize glucose, which would be preferably metabolized by the Entner-Dudoroff pathway [38]. In X. fastidiosa, all genes of the Entner-Dudoroff pathway are encoded by a single operon, which encompasses ORFs Xf1061 to Xf1065, but we did not observe overexpression of any such genes in either of the media, even in 3G10R, which has glucose as the sole carbon source.The difference in carbon source also seems to be important in determining the expression of genes associated with other aspects of the cellular metabolism, such as aminoacid biosynthesis (in 3G10R), as opposed to proteolytic enzymes (in PW). Interestingly, the coordinated upregulation of proteolytic enzymes is indicative thatX. fastidiosa cells, like lactic acid bacteria, have developed an efficient mechanism dedicated to process extra cellular proteins as a major way to obtain amino acids from exogenous sources [39]. This idea is also consistent with the elevated growth rates observed with cells grown in PW, a significantly rich medium, which is based on relatively high concentrations of protein hydrolisates, such as tryptone and peptone [11].In spite of providing more adequate nutritional conditions to sustain continued growth of fastidious microorganisms, complex media are not likely to resemble the harsh nutritional conditions found in xylem sap. Since 3G10R does not receive nutrients from any complex source, it is likely to be much more restricted in nutrient availability [16]. Moreover, this formulation incorporates a few important chemical characteristics that resemble xylem composition of plants known to be infected by X. fastidiosa, such as the use of glucose as a major carbon source [22–24] and the presence of L-glutamine, which is the most abundant amino acid detected in the sap of grapevines [25, 26] and seems to be essential for in vitro growth of X. fastidiosa cells [11, 40]. The antioxidant tripeptide glutathione (GSH) has also been detected in the composition of xylem fluid of poplar and spruce trees [41, 42] and is present in the composition of 3G10R at a similar concentration [16].The presence of glucose seems to be an important characteristic of 3G10R in resembling xylem, since this metabolite has already been identified in the chemistry composition of xylem fluid from many plant species, such as grapevine [22], maize [43], cabbage [44], poplar [24] and oak [23], among others [45]. However, the exact glucose concentration found in the xylem sap of different plants has been shown to vary significantly, depending on the species, genotype, season, time of day, age of plants and nutritional status. In poplar trees, such concentration has been shown to range from 0.2 to 15 mM [24], although there have been reports of this nutrient at <50 μM concentration in the xylem of grapevines (a typical X. fastidiosa host) [16]. Thus, the ~10 mM glucose concentration present in 3G10R might be higher than the concentration typically encountered by X. fastidiosa cells during the process of plant infection and colonization.Although glucose is generally viewed as an energy source for growing microorganisms, this substance has also been shown to act as a precursor for the biosynthesis of several bacterial cell wall components and exopolysaccharides (EPSs), which have been proposed to act as virulence factors inX. fastidiosa and many other pathogenic bacteria ([46–48], reviewed in [4]). Moreover, increased production of EPS is one major characteristic of X. fastidiosa cells freshly isolated from infected plants and such primarily isolated cells have been shown to be more effective in the process of plant colonization, when compared to cells submitted to continued growth in PW [49]. Coincidentally, while growing in 3G10R, X. fastidiosa cells have also been shown to synthesize increased amounts of EPS, leading to more intense biofilm formation [16]. It has even been proposed that the preferential use of glucose to drive EPS synthesis could be an explanation to the fastidious growth of X. fastidiosa cells, especially in 3G10R, where these molecules are expected to act as the major source of energy as well [16]. Interestingly, when X. fastidiosa cells are grown in this medium, we observed increased expression of xanA (ORF Xf0260), which encodes a phosphoglucomutase that converts glucose 6-P into glucose 1-P, which in turn, acts as a precursor of UDP-Glucose and UDP-Galactose, which are involved in the biosynthesis of different types of EPS [50]. Moreover, it has already been shown that increased expression of phosphoglucomutase can lead to an increase in the production of EPS in Lactococus lactis [51].EPS production is one of the most important aspects of biofilm formation, which is believed to be an important pathogenicity factor inX. fastidiosacells [52]. Other adhesion factors have been detected as preferentially expressed in 3G10R, which might be directly correlated with the more intense cellular aggregation and biofilm formation observed in this medium [16]. One of these putative adhesion factors is represented by ORF Xf0487, which encodes a 20 kDa fimbrillin subunit of bacterial fimbreae, and may be involved in bacterial adherence and invasion [53]. Pili and fimbreae have been implicated in plant infection and migration via a twitching motility mechanism that seems to be of paramount importance to the colonization process of X. fastidiosa [54]. Another important component of the cellular outer membrane structure that has been shown to be upregulated in 3G10R is the hsf gene (ORF Xf1529), which encodes a surface fibril that belongs to a family of high molecular weight autotransporter adhesins [55]. This protein has been originally characterized as an important virulence factor from Haemophilus influenzae type b, which causes meningitis and other serious invasive human diseases. In this bacterium, the Hsf protein has been shown to form trimeric fiber-like structures on the bacterial surface that mediate adhesion to epithelial cells [56]. Hsf is also suspected to act as a virulence factor in X. fastidiosa, since overexpression of this protein occurs in X. fastidiosa cells that display higher infective capacity, as well as in bacteria directly isolated from infected plants [17, 49].Three bacteriocin genes (Xf0262, Xf0263 and Xf0264) have been found to be overexpressed in 3G10R-cultivated cells, suggesting that increased production of such molecules might be important to X. fastidiosa cells in competing with other endophytic bacteria within the xylem [57]. These molecules belong to a class of structurally related proteins that kill target cells by membrane permeabilization. Some of them have been known to kill different types of bacteria, constituting a strategic advantage for microorganisms that colonize highly competitive environments [58]. Although little is known about the X. fastidiosa bacteriocins so far, it is interesting to verify that the bacteriocin encoded by Xf0263 has also been identified as overexpressed in X. fastidiosa cells that display higher infective capacity, as well as in bacteria directly isolated from infected plants [17, 49], while the proteins encoded by Xf0262 and Xf0264 are induced in response to glucose [59].Although we are aware that defined media, like 3G10R, do not constitute a perfect simulation of the environment inhabited by xylem-dwelling endophytes, this formulation has clearly incorporated some important chemical aspects of xylem fluid composition, which induce transcriptional activation of some putative pathogenicity-associated genes inX. fastidiosa cells. Moreover, some of these genes have also been shown to be specifically upregulated in cells directly isolated from infected plants, as well as in freshly isolated X. fastidiosa cultures, which are known to display a higher infective capacity. The dependence of aggregation and biofilm formation on the nutrient composition of xylem fluid suggests that xylem chemistry is important in resistance/susceptibility to disease [27, 60, 61]. Thus, the transcriptome profile of X. fastidiosa cells grown in xylem-based chemistry media is more likely to represent the metabolome of X. fastidiosa cells in planta, reinforcing the idea that such media formulations should be preferred for metabolic studies of this phytopathogen.



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*Source: 781365-2010-06-13.xml*