Xylella fastidiosa subsp. pauca Strains Fb7 and 9a5c from Citrus Display Differential Behavior, Secretome, and Plant Virulence.

Xylella fastidiosa colonizes the xylem of various cultivated and native plants worldwide. Citrus production in Brazil has been seriously affected, and major commercial varieties remain susceptible to Citrus Variegated Chlorosis (CVC). Collective cellular behaviors such as biofilm formation influence virulence and insect transmission of X. fastidiosa. The reference strain 9a5c produces a robust biofilm compared to Fb7 that remains mostly planktonic, and both were isolated from symptomatic citrus trees. This work deepens our understanding of these distinct behaviors at the molecular level, by comparing the cellular and secreted proteomes of these two CVC strains. Out of 1017 identified proteins, 128 showed differential abundance between the two strains. Different protein families were represented such as proteases, hemolysin-like proteins, and lipase/esterases, among others. Here we show that the lipase/esterase LesA is among the most abundant secreted proteins of CVC strains as well, and demonstrate its functionality by complementary activity assays. More severe symptoms were observed in Nicotiana tabacum inoculated with strain Fb7 compared to 9a5c. Our results support that systemic symptom development can be accelerated by strains that invest less in biofilm formation and more in plant colonization. This has potential application in modulating the bacterial-plant interaction and reducing disease severity.

BigR is a sulfide sensor that regulates a sulfur transferase/dioxygenase required for aerobic respiration of plant bacteria under sulfide stress.

To cope with toxic levels of H2S, the plant pathogens Xylella fastidiosa and Agrobacterium tumefaciens employ the bigR operon to oxidize H2S into sulfite. The bigR operon is regulated by the transcriptional repressor BigR and it encodes a bifunctional sulfur transferase (ST) and sulfur dioxygenase (SDO) enzyme, Blh, required for H2S oxidation and bacterial growth under hypoxia. However, how Blh operates to enhance bacterial survival under hypoxia and how BigR is deactivated to derepress operon transcription is unknown. Here, we show that the ST and SDO activities of Blh are in vitro coupled and necessary to oxidize sulfide into sulfite, and that Blh is critical to maintain the oxygen flux during A. tumefaciens respiration when oxygen becomes limited to cells. We also show that H2S and polysulfides inactivate BigR leading to operon transcription. Moreover, we show that sulfite, which is produced by Blh in the ST and SDO reactions, is toxic to Citrus sinensis and that X. fastidiosa-infected plants accumulate sulfite and higher transcript levels of sulfite detoxification enzymes, suggesting that they are under sulfite stress. These results indicate that BigR acts as a sulfide sensor in the H2S oxidation mechanism that allows pathogens to colonize plant tissues where oxygen is a limiting factor.

Caracterização bioquímica e funcional da proteína XadA3 de Xylella fastidiosa / Biochemistry and functional characterization of Xylella fastidiosa XadA3 adhesin

Gram-negativas e é utilizado por diversos patógenos para colonizar seus hospedeiros, sendo o primeiro passo do processo de desenvolvimento do biolfilme. Uma variedade de apêndices celulares e proteínas está envolvida na adesão bacteriana, tais como pili, fimbrias, adesinas fimbriais e afimbriais. O fitopatógeno Xylella fastidiosa, agente causal de importantes doenças como a doença de Pierce de videiras, a clorose variegada dos citros e a síndrome do rápido declínio de oliveiras, possui em sua superfície várias dessas estruturas que são potencialmente responsáveis pela colonização eficiente de insetos-vetores e plantas hospedeiras. Entre as adesinas afimbriais codificadas no genoma dessa bactéria, três XadA (XadA1, Hsf/XadA2 e XadA3) são classificadas como autotransportadores triméricos. Dados da literatura sugerem que XadA1 e XadA2 são importantes para a formação do biofilme, porém a função de XadA3 ainda não havia sido investigada. Nesse trabalho, tivemos como objetivo caracterizar bioquímica e funcionalmente a proteína XadA3 e obter informações adicionais sobre o papel desempenhado por XadA1 e XadA2 na adesão e virulência de X. fastidiosa. Utilizando imunodetecção com um anticorpo policlonal anti-XadA3 por nós obtido, demonstramos que essa proteína localiza-se na superfície bacteriana e medeia a adesão intercelular. A caracterização dos fenótipos de mutantes de deleção de cada um dos genes das adesinas XadA revelou que o mutante ΔxadA3 tem reduzida capacidade de agregação celular e formação de biofilme quando comparado tanto aos mutantes ΔxadA1 e ΔxadA2 como à cepa selvagem Temecula. A deleção dos genes xadA afeta marginalmente o perfil de expressão gênica global avaliado através de RNAseq das cepas mutantes comparativamente à cepa selvagem, porém destaca-se, nas cepas mutantes, o aumento nos níveis dos transcritos de lipases/esterases. Já foi descrito que essas enzimas parecem atuar na degradação do tecido vegetal associada aos sintomas da doença de Pierce de videiras. A deleção de xadA3 resulta em um fenótipo de hipervirulência em videiras, mas também de deficiência de transmissão pelo inseto-vetor. O conjunto dos resultados obtidos nesse trabalho evidenciam o importante papel desempenhado pelas adesinas XadAs, particularmente XadA3, na adesão intercelular, no desenvolvimento do biofilme e na virulência de X. fastidiosa

Adhesion is a widely conserved mechanism of virulence among Gram-negative bacteria that is used by several pathogens to colonize their hosts, being the first step in biolfilm development. A variety of appendages and proteins are involved in bacterial adhesion, such as pili, fimbriae, fimbrial and afimbrials adhesins. The phytopathogen Xylella fastidiosa, causal agent of important diseases such as Pierce's disease of grapevines, citrus variegated chlorosis and olive quick decline syndrome, harbours on its surface several of these structures that are potentially responsible for efficient colonization of insect vectors and plant hosts. Among the afimbrial adhesins encoded in the genome of this bacterium, three XadAs (XadA1, Hsf/XadA2 and XadA3) are classified as trimeric autotransporters. Data from the literature suggest that XadA1 and XadA2 are important for biofilm formation, but XadA3 function has not been yet investigated. In this work, we aimed to biochemically and functionally characterize the XadA3 protein and gather additional information about the role played by XadA1 and XadA2 in X. fastidiosa adhesion and virulence. Using immunodetection with a polyclonal anti-XadA3 antibody, we have demonstrated that this protein localizes to the bacterial surface and mediates intercellular adhesion. Phenotypic characterization of the deletion mutants of XadA adhesins encoded genes revealed that the ΔxadA3 mutant has reduced cell aggregation capacity and biofilm formation when compared to both ΔxadA1 and ΔxadA2 mutants as well as to Temecula wild type strain. Deletion of the xadA genes marginally affects the global gene expression profile assessed by RNA-seq of the mutant strains compared to the wild-type strain, eventhough an increase in lipase/esterase transcripts levels was observed in the mutant strains. It has been reported that these enzymes appear to participate in the degradation of plant tissue that is associated with symptoms of Pierce's disease of grapevines. The deletion of xadA3 results in a phenotype of hypervirulence in grapevines but also of deficiency in insect-vector transmission. The results obtained in this work evidenced the important role played by XadAs adhesins, particularly XadA3, in X. fastidiosa intercellular adhesion, biofilm development and virulence

Ectopic Expression of Xylella fastidiosa rpfF Conferring Production of Diffusible Signal Factor in Transgenic Tobacco and Citrus Alters Pathogen Behavior and Reduces Disease Severity.

The pathogenicity of Xylella fastidiosa is associated with its ability to colonize the xylem of host plants. Expression of genes contributing to xylem colonization are suppressed, while those necessary for insect vector acquisition are increased with increasing concentrations of diffusible signal factor (DSF), whose production is dependent on RpfF. We previously demonstrated that transgenic citrus plants ectopically expressing rpfF from a citrus strain of X. fastidiosa subsp. pauca exhibited less susceptibility to Xanthomonas citri subsp. citri, another pathogen whose virulence is modulated by DSF accumulation. Here, we demonstrate that ectopic expression of rpfF in both transgenic tobacco and sweet orange also confers a reduction in disease severity incited by X. fastidiosa and reduces its colonization of those plants. Decreased disease severity in the transgenic plants was generally associated with increased expression of genes conferring adhesiveness to the pathogen and decreased expression of genes necessary for active motility, accounting for the reduced population sizes achieved in the plants, apparently by limiting pathogen dispersal through the plant. Plant-derived DSF signal molecules in a host plant can, therefore, be exploited to interfere with more than one pathogen whose virulence is controlled by DSF signaling.

Crystal structure of a small heat-shock protein from Xylella fastidiosa reveals a distinct high-order structure.

Citrus variegated chlorosis is a disease that attacks economically important citrus plantations and is caused by the plant-pathogenic bacterium Xylella fastidiosa. In this work, the structure of a small heat-shock protein from X. fastidiosa (XfsHSP17.9) is reported. The high-order structures of small heat-shock proteins from other organisms are arranged in the forms of double-disc, hollow-sphere or spherical assemblies. Unexpectedly, the structure reported here reveals a high-order architecture forming a nearly square cavity.

Characterization of the TolB-Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process.

The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development.

Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach.

The lack of efficient refolding methodologies must be overcome to take full advantage of the fact that bacteria express high levels of aggregated recombinant proteins. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, dissociating aggregates, which makes HHP a useful tool to solubilize proteins for subsequent refolding. A process of refolding was set up by using as a model TsnC, a thioredoxin that catalyzes the disulfide reduction to a dithiol, a useful indication of biological activity. The inclusion bodies (IB) were dissociated at 2.4 kbar. The effect of incubation of IB suspensions at 1-800 bar, the guanidine hydrochloride concentration, the oxidized/reduced glutathione (GSH/GSSG) ratios, and the additives in the refolding buffer were analyzed. To assess the yields of fully biologically active protein obtained for each tested condition, it was crucial to analyze both the TsnC solubilization yield and its enzymatic activity. Application of 2.4 kbar to the IB suspension in the presence of 9 mM GSH, 1mM GSSG, 0.75 M guanidine hydrochloride, and 0.5M arginine with subsequent incubation at 1 bar furnished high refolding yield (81%). The experience gained in this study shall help to establish efficient HHP-based protein refolding processes for other proteins.

Surface physicochemical properties at the micro and nano length scales: role on bacterial adhesion and Xylella fastidiosa biofilm development.

The phytopathogen Xylella fastidiosa grows as a biofilm causing vascular occlusion and consequently nutrient and water stress in different plant hosts by adhesion on xylem vessel surfaces composed of cellulose, hemicellulose, pectin and proteins. Understanding the factors which influence bacterial adhesion and biofilm development is a key issue in identifying mechanisms for preventing biofilm formation in infected plants. In this study, we show that X. fastidiosa biofilm development and architecture correlate well with physicochemical surface properties after interaction with the culture medium. Different biotic and abiotic substrates such as silicon (Si) and derivatized cellulose films were studied. Both biofilms and substrates were characterized at the micro- and nanoscale, which corresponds to the actual bacterial cell and membrane/ protein length scales, respectively. Our experimental results clearly indicate that the presence of surfaces with different chemical composition affect X. fastidiosa behavior from the point of view of gene expression and adhesion functionality. Bacterial adhesion is facilitated on more hydrophilic surfaces with higher surface potentials; XadA1 adhesin reveals different strengths of interaction on these surfaces. Nonetheless, despite different architectural biofilm geometries and rates of development, the colonization process occurs on all investigated surfaces. Our results univocally support the hypothesis that different adhesion mechanisms are active along the biofilm life cycle representing an adaptation mechanism for variations on the specific xylem vessel composition, which the bacterium encounters within the infected plant.

Small-angle X-ray scattering and in silico modeling approaches for the accurate functional annotation of an LysR-type transcriptional regulator.

Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogen responsible for economically relevant crop diseases. Its genome was thus sequenced in an effort to characterize and understand its metabolism and pathogenic mechanisms. However, the assignment of the proper functions to the identified open reading frames (ORFs) of this pathogen was impaired due to a lack of sequence similarity in the databases. In the present work, we used small-angle X-ray scattering and in silico modeling approaches to characterize and assign a function to a predicted LysR-type transcriptional regulator in the X. fastidiosa (XfLysRL) genome. XfLysRL was predicted to be a homologue of BenM, which is a transcriptional regulator involved in the degradation pathway of aromatic compounds. Further functional assays confirmed the structural prediction because we observed that XfLysRL interacts with benzoate and cis,cis-muconic acid (also known as 2E,4E-hexa-2,4-dienedioic acid; hereafter named muconate), both of which are co-factors of BenM. In addition, we showed that the XfLysRL protein is differentially expressed during the different stages of X. fastidiosa biofilm formation and planktonic cell growth, which indicates that its expression responds to a cellular signal that is likely related to the aromatic compound degradation pathway. The assignment of the proper function to a protein is a key step toward understanding the cellular metabolic pathways and pathogenic mechanisms. In the context of X. fastidiosa, the characterization of the predicted ORFs may lead to a better understanding of the cellular pathways that are linked to its bacterial pathogenicity.

On the role of extracellular polymeric substances during early stages of Xylella fastidiosa biofilm formation.

The structural integrity and protection of bacterial biofilms are intrinsically associated with a matrix of extracellular polymeric substances (EPS) produced by the bacteria cells. However, the role of these substances during biofilm adhesion to a surface remains largely unclear. In this study, the influence of EPS on Xylella fastidiosa biofilm formation was investigated. This bacterium is associated with economically important plant diseases; it presents a slow growth rate and thus allows us to pinpoint more precisely the early stages of cell-surface adhesion. Scanning electron microscopy and atomic force microscopy show evidence of EPS production in such early stages and around individual bacteria cells attached to the substrate surface even a few hours after inoculation. In addition, EPS formation was investigated via attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (FTIR). To this end, X. fastidiosa cells were inoculated within an ATR liquid cell assembly. IR-ATR spectra clearly reveal EPS formation already during the early stages of X. fastidiosa biofilm formation, thereby providing supporting evidence for the hypothesis of the relevance of the EPS contribution to the adhesion process.

Global expression profile of biofilm resistance to antimicrobial compounds in the plant-pathogenic bacterium Xylella fastidiosa reveals evidence of persister cells.

Investigations of biofilm resistance response rarely focus on plant-pathogenic bacteria. Since Xylella fastidiosa is a multihost plant-pathogenic bacterium that forms biofilm in the xylem, the behavior of its biofilm in response to antimicrobial compounds needs to be better investigated. We analyzed here the transcriptional profile of X. fastidiosa subsp. pauca in response to inhibitory and subinhibitory concentrations of copper and tetracycline. Copper-based products are routinely used to control citrus diseases in the field, while antibiotics are more widely used for bacterial control in mammals. The use of antimicrobial compounds triggers specific responses to each compound, such as biofilm formation and phage activity for copper. Common changes in expression responses comprise the repression of genes associated with metabolic functions and movement and the induction of toxin-antitoxin systems, which have been associated with the formation of persister cells. Our results also show that these cells were found in the population at a ca. 0.05% density under inhibitory conditions for both antimicrobial compounds and that pretreatment with subinhibitory concentration of copper increases this number. No previous report has detected the presence of these cells in X. fastidiosa population, suggesting that this could lead to a multidrug tolerance response in the biofilm under a stressed environment. This is a mechanism that has recently become the focus of studies on resistance of human-pathogenic bacteria to antibiotics and, based on our data, it seems to be more broadly applicable.

Plant pathogenic bacteria utilize biofilm growth-associated repressor (BigR), a novel winged-helix redox switch, to control hydrogen sulfide detoxification under hypoxia.

Winged-helix transcriptional factors play important roles in the control of gene expression in many organisms. In the plant pathogens Xylella fastidiosa and Agrobacterium tumefaciens, the winged-helix protein BigR, a member of the ArsR/SmtB family of metal sensors, regulates transcription of the bigR operon involved in bacterial biofilm growth. Previous studies showed that BigR represses transcription of its own operon through the occupation of the RNA polymerase-binding site; however, the signals that modulate its activity and the biological function of its operon are still poorly understood. Here we show that although BigR is a homodimer similar to metal sensors, it functions as a novel redox switch that derepresses transcription upon oxidation. Crystal structures of reduced and oxidized BigR reveal that formation of a disulfide bridge involving two critical cysteines induces conformational changes in the dimer that remarkably alter the topography of the winged-helix DNA-binding interface, precluding DNA binding. This structural mechanism of DNA association-dissociation is novel among winged-helix factors. Moreover, we demonstrate that the bigR operon is required for hydrogen sulfide detoxification through the action of a sulfur dioxygenase (Blh) and sulfite exporter. As hydrogen sulfide strongly inhibits cytochrome c oxidase, it must be eliminated to allow aerobic growth under low oxygen tension, an environmental condition found in bacterial biofilms, xylem vessels, and root tissues. Accordingly, we show that the bigR operon is critical to sustain bacterial growth under hypoxia. These results suggest that BigR integrates the transcriptional regulation of a sulfur oxidation pathway to an oxidative signal through a thiol-based redox switch.

Characterization of an oxidative stress response regulator, homologous to Escherichia coli OxyR, from the phytopathogen Xylella fastidiosa.

The OxyR oxidative stress transcriptional regulator is a DNA-binding protein that belongs to the LysR-type transcriptional regulators (LTTR) family. It has the ability to sense oxidative species inside the cell and to trigger the cell's response, activating the transcription of genes involved in scavenging oxidative species. In the present study, we have overexpressed, purified and characterized the predicted OxyR homologue (orf xf1273) of the phytopathogen Xylella fastidiosa. This bacterium is the causal agent of citrus variegated chlorosis (CVC) disease caused by the 9a5c strain, resulting in economic and social losses. The secondary structure of the recombinant protein was analyzed by circular dichroism. Gel filtration showed that XfoxyR is a dimer in solution. Gel shift assays indicated that it does bind to its own predicted promoter under in vitro conditions. However, considering our control experiment we cannot state that this interaction occurs in vivo. Functional complementation assays indicated that xfoxyR is able to restore the oxidative stress response in an oxyr knockout Escherichia coli strain. These results show that the predicted orfxf1273 codes for a transcriptional regulator, homologous to E. coli OxyR, involved in the oxidative stress response. This may be important for X. fastidiosa to overcome the defense mechanisms of its host during the infection and colonization processes.

The usage of codons which are similar to stop codons in the genomes of Xylella fastidiosa and Xanthomonas citri.

During the evolution of living organisms, a natural selection event occurs toward the optimization of their genomes regarding the usage of codons. During this process which is known as codon bias, a set of preferred codons is naturally defined in the genome of a given organism, since there are 61 possible codons (plus 3 stop codons) to 20 amino acids. Such event leads to optimization of metabolic cellular processes such as translational efficiency, RNA stability and energy saving. Although we know why, we do not know how exactly a set of preferred codons for each amino acid is defined for a given genome considering that the usage frequency of each synonymous codons is peculiar to each organism. In order to help answering this question, we analyzed the usage frequency of codons which are similar to stop codons, since a minor mutation on these codons may lead to a stop codon into the open reading frame compromising the protein expression as a result. We found a reduced use of those codons in Xanthomomas axonopodis pv. citri which presents an optimized genome regarding codon usage. On the other hand, such codons are more often used in Xylella fastidiosa, which does not seem to have established codon preferences as previously shown. Our results support that a set of preferred codons is not randomly selected and propose new ideas to the field warranting further experiments in this regard.

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon.

BACKGROUND: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. RESULTS: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ54-dependent manner. A more complete picture of the σ54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ54-dependent promoters, using a position weight matrix approach. One of these σ54-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ54-dependent promoter. CONCLUSIONS: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ54 regulon.

Diversity of endophytic yeasts from sweet orange and their localization by scanning electron microscopy.

Endophytes are microorganisms that colonize plant tissues internally without causing harm to the host. Despite the increasing number of studies on sweet orange pathogens and endophytes, yeast has not been described as a sweet orange endophyte. In the present study, endophytic yeasts were isolated from sweet orange plants and identified by sequencing of internal transcribed spacer (ITS) rRNA. Plants sampled from four different sites in the state of São Paulo, Brazil exhibited different levels of CVC (citrus variegated chlorosis) development. Three citrus endophytic yeasts (CEYs), chosen as representative examples of the isolates observed, were identified as Rhodotorula mucilaginosa, Pichia guilliermondii and Cryptococcus flavescens. These strains were inoculated into axenic Citrus sinensis seedlings. After 45 days, endophytes were re-isolated in populations ranging from 10(6) to 10(9) CFU/g of plant tissue, but, in spite of the high concentrations of yeast cells, no disease symptoms were observed. Colonized plant material was examined by scanning electron microscopy (SEM), and yeast cells were found mainly in the stomata and xylem of plants, reinforcing their endophytic nature. P. guilliermondii was isolated primarily from plants colonized by the causal agent of CVC, Xylella fastidiosa. The supernatant from a culture of P. guilliermondii increased the in vitro growth of X. fastidiosa, suggesting that the yeast could assist in the establishment of this pathogen in its host plant and, therefore, contribute to the development of disease symptoms.

Functional and small-angle X-ray scattering studies of a new stationary phase survival protein E (SurE) from Xylella fastidiosa--evidence of allosteric behaviour.

The genome data of bacterium Xylella fastidiosa strain 9a5c has identified several orfs related to its phytopathogenic adaptation and survival. Among these genes, the surE codifies a survival protein E (XfSurE) whose function is not so well understood, but functional assays in Escherichia coli revealed nucleotidase and exopolyphosphate activity. In the present study, we report the XfSurE protein overexpression in E. coli and its purification. The overall secondary structure was analyzed by CD. Small-angle X-ray scattering and gel filtration techniques demonstrated that the oligomeric state of the protein in solution is a tetramer. In addition, functional kinetics experiments were carried out with several monophosphate nucleoside substrates and revealed a highly positive cooperativity. An allosteric mechanism involving torsion movements in solution is proposed to explain the cooperative behaviour of XfSurE. This is the first characterization of a SurE enzyme from a phytopathogen organism and, to our knowledge, the first solution structure of a SurE protein to be described.

The iron stimulon of Xylella fastidiosa includes genes for type IV pilus and colicin V-like bacteriocins.

Xylella fastidiosa is the etiologic agent of a wide range of plant diseases, including citrus variegated chlorosis (CVC), a major threat to citrus industry. The genomes of several strains of this phytopathogen were completely sequenced, enabling large-scale functional studies. DNA microarrays representing 2,608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c were used to investigate transcript levels during growth with different iron availabilities. When treated with the iron chelator 2,2'-dipyridyl, 193 CDS were considered up-regulated and 216 were considered down-regulated. Upon incubation with 100 microM ferric pyrophosphate, 218 and 256 CDS were considered up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription-quantitative PCR. Several CDS involved with regulatory functions, pathogenicity, and cell structure were modulated under both conditions assayed, suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to functions of pili/fimbriae. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. The promoter regions of the strain 9a5c genome were screened for putative Fur boxes, and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X. fastidiosa, and they present novel evidence for iron regulation of pathogenicity determinants.

Role of sigma54 in the regulation of genes involved in type I and type IV pili biogenesis in Xylella fastidiosa.

The phytopathogen Xylella fastidiosa produces long type IV pili and short type I pili involved in motility and adhesion. In this work, we have investigated the role of sigma factor sigma(54) (RpoN) in the regulation of fimbrial biogenesis in X. fastidiosa. An rpoN null mutant was constructed from the non-pathogenic citrus strain J1a12, and microarray analyses of global gene expression comparing the wild type and rpoN mutant strains showed few genes exhibiting differential expression. In particular, gene pilA1 (XF2542), which encodes the structural pilin protein of type IV pili, showed decreased expression in the rpoN mutant, whereas two-fold higher expression of an operon encoding proteins of type I pili was detected, as confirmed by quantitative RT-PCR (qRT-PCR) analysis. The transcriptional start site of pilA1 was determined by primer extension, downstream of a sigma(54)-dependent promoter. Microarray and qRT-PCR data demonstrated that expression of only one of the five pilA paralogues, pilA1, was significantly reduced in the rpoN mutant. The rpoN mutant made more biofilm than the wild type strain and presented a cell-cell aggregative phenotype. These results indicate that sigma(54) differentially regulates genes involved in type IV and type I fimbrial biogenesis, and is involved in biofilm formation in X. fastidiosa.

Absence of classical heat shock response in the citrus pathogen Xylella fastidiosa.

The fastidious bacterium Xylella fastidiosa is associated with important crop diseases worldwide. We have recently shown that X. fastidiosa is a peculiar organism having unusually low values of gene codon bias throughout its genome and, unexpectedly, in the group of the most abundant proteins. Here, we hypothesized that the lack of codon usage optimization in X. fastidiosa would incapacitate this organism to undergo quick and massive changes in protein expression as occurs in a classical stress response. Proteomic analysis of the response to heat stress in X. fastidiosa revealed that no changes in protein expression can be detected. Moreover, stress-inducible proteins identified in the closely related citrus pathogen Xanthomonas axonopodis pv citri were found to be constitutively expressed in X. fastidiosa. These proteins have extremely high codon bias values in the X. citri and other well-studied organisms, but low values in X. fastidiosa. Because biased codon usage is well known to correlate to the rate of protein synthesis, we speculate that the peculiar codon bias distribution in X. fastidiosa is related to the absence of a classical stress response, and, probably, alternative strategies for survival of X. fastidiosa under stressfull conditions.