Bioremediation of the herbicide glyphosate in polluted soils by plant-associated microbes.

Most productive lands worldwide base their crop production on the use of glyphosate (GLY)-resistant plants, and consequently, widespread use of this herbicide has led to environmental issues that need to be solved. Soil bioremediation technologies based on degradation of GLY by microorganisms are strategies that have been considered useful to solve this environmental problem. Recently, a further step has been taken considering the use of bacteria that interact with plants, either alone or both bacteria and plant together, for the removal of GLY herbicide. Plant-interacting microorganisms with plant growth-promoting traits can also enhance plant growth and contribute to successful bioremediation strategies.

Xanthomonas natriuretic peptide is recognized by the Arabidopsis natriuretic peptide receptor 1 and through this interaction triggers similar plant responses to its plant counterpart.

Plant natriuretic peptides (PNPs) are hormone peptides that participate in the regulation of ions and water homeostasis in plants. Xanthomonas citri subsp. citri (Xcc) the causal agent of citrus canker disease also possesses a PNP-like peptide (XacPNP). This peptide, similarly to AtPNP-A, the most studied PNP from Arabidopsis thaliana, causes stomatal aperture and enhances photosynthetic efficiency in plant leaves. Thus, the function that has been attributed to XacPNP is to contribute to maintain photosynthetic efficiency and water homeostasis in plant tissue during the infection process, to create favorable conditions for biotrophic pathogens survival. A PNP receptor (AtPNP-R1) for AtPNP-A has been identified and the AtPNP-A activity in regulation of water homeostasis has been observed to depend on the presence of AtPNP-R1. Here, we demonstrated that both AtPNP-A and XacPNP require the presence of AtPNP-R1 to induce plant stomatal aperture. Also, less necrotic tissue was found in infections with pathogens expressing XacPNP and this was dependent on the presence of AtPNP-R1, suggesting that XacPNP interacts with this receptor to exert its function. Finally, we confirmed that AtPNP-A and XacPNP interact with AtPNP-R1 in planta, which support the idea that XacPNP triggers similar plant responses to its plant counterpart.

Cyclic di-GMP Signaling Links Biofilm Formation and Mn(II) Oxidation in Pseudomonas resinovorans.

Bioaugmentation of biological sand filters with Mn(II)-oxidizing bacteria (MOB) is used to increase the efficiency of Mn removal from groundwater. While the biofilm-forming ability of MOB is important to achieve optimal Mn filtration, the regulatory link between biofilm formation and Mn(II) oxidation remains unclear. Here, an environmental isolate of Pseudomonas resinovorans strain MOB-513 was used as a model to investigate the role of c-di-GMP, a second messenger crucially involved in the regulation of biofilm formation by Pseudomonas, in the oxidation of Mn(II). A novel role for c-di-GMP in the upregulation of Mn(II) oxidation through induction of the expression of manganese-oxidizing peroxidase enzymes was revealed. MOB-513 macrocolony biofilms showed a strikingly stratified pattern of biogenic Mn oxide (BMnOx) accumulation in a localized top layer. Remarkably, elevated cellular levels of c-di-GMP correlated not only with increased accumulation of BMnOx in the same top layer but also with the appearance of a second BMnOx stratum in the bottom region of macrocolony biofilms, and the expression of mop genes correlated with this pattern. Proteomic analysis under Mn(II) conditions revealed changes in the abundance of a PilZ domain protein. Subsequent analyses supported a model in which this protein sensed c-di-GMP and affected a regulatory cascade that ultimately inhibited mop gene expression, providing a molecular link between c-di-GMP signaling and Mn(II) oxidation. Finally, we observed that high c-di-GMP levels were correlated with higher lyophilization efficiencies and higher groundwater Mn(II) oxidation capacities of freeze-dried bacterial cells, named lyophiles, showing the biotechnological relevance of understanding the role of c-di-GMP in MOB-513. IMPORTANCE The presence of Mn(II) in groundwater, a common source of drinking water, is a cause of water quality impairment, interfering with its disinfection, causing operation problems, and affecting human health. Purification of groundwater containing Mn(II) plays an important role in environmental and social safety. The typical method for Mn(II) removal is based on bacterial oxidation of metals to form insoluble oxides that can be filtered out of the water. Evidence of reducing the start-up periods and enhancing Mn removal efficiencies through bioaugmentation with appropriate biofilm-forming and MOB has emerged. As preliminary data suggest a link between these two phenotypes in Pseudomonas strains, the need to investigate the underlying regulatory mechanisms is apparent. The significance of our research lies in determining the role of c-di-GMP for increased biofilm formation and Mn(II)-oxidizing capabilities in MOB, which will allow the generation of super-biofilm-elaborating and Mn-oxidizing strains, enabling their implementation in biotechnological applications.

Manganese oxidation counteracts the deleterious effect of low temperatures on biofilm formation in Pseudomonas sp. MOB-449.

Mn removal from groundwater by biological sand filter technology is negatively impacted by low temperatures in winter periods. Therefore, the need to study Mn(II)-oxidizing bacteria (MOB) having the potential to oxidize Mn(II) and form biofilms at low temperatures is imperative. These MOB can have potential as inocula for sand filter bioaugmentation strategies to optimize Mn removal during winter periods. We previously showed that a Pseudomonas sp. MOB-449 (MOB-449), isolated from a Mn biofilter, oxidizes Mn(II) in a biofilm-dependent way at low temperatures. In this work, MOB-449 Mn(II) oxidation and growth capacities were evaluated under planktonic and biofilm conditions at different temperatures. At 18°C, MOB-449 showed enhanced biofilm formation due to the addition of Mn(II) to the medium correlating with Mn(II) oxidation, compared to biofilms grown in control medium. Moreover, this enhancement on biofilm formation due to the addition of Mn(II) was only observed at 18°C. At this temperature, Mn(II) oxidation in membrane fractions collected from biofilms was induced by uncoupling oxidative phosphorylation from the electron transport chain with 2,4-Dinitrophenol. In Pseudomonas, a role for c-type cytochrome in Mn(II) oxidation has been demonstrated. Accordingly, transcriptional profiles of all terminal oxidases genes found in MOB-449 showed an induction of cytochrome c terminal oxidases expression mediated by Mn(II) oxidation at 18°C. Finally, heme peroxidase activity assays and MS analysis revealed that PetC, a cytochrome c5, and also CcmE, involved in the cytochrome c biogenesis machinery, are induced at 18°C only in the presence of Mn(II). These results present evidence supporting that cytochromes c and also the cytochrome c terminal oxidases are activated at low temperatures in the presence of Mn(II). Overall, this work demonstrate that in MOB-449 Mn(II) oxidation is activated at low temperatures to gain energy, suggesting that this process is important for survival under adverse environmental conditions and contributing to the understanding of the physiological role of bacterial Mn(II) oxidation.

Bacterial isolates from Argentine Pampas and their ability to degrade glyphosate.

Glyphosate is a synthetic phosphonate compound characterized by a carbon­phosphorus bond. Glyphosate based herbicides (GBH) are widely distributed in most of the economically productive lands in which crop production is mainly based on glyphosate-resistant genetically modified plants. Naturally, glyphosate is remediated by soil microorganisms, which accelerate its degradation. Technology based on microorganisms is considered highly efficient, low-cost and eco-friendly to remediate contaminated environments, denoting the importance of characterizing new bacterial strains able to degrade glyphosate to perform its bioremediation. In this work, 13 different bacterial strains able to grow in GBH as only phosphorous source were isolated from different environmental samples from the Argentine vastly productive glyphosate-resistant soybean crop area. These strains were identified and they belong to the genera Acinetobacter, Achromobacter, Agrobacterium, Ochrobactrum, Pantoea and Pseudomonas. Their ability to grow and consume GBH, glyphosate or the aminomethylphosphonic acid (AMPA), another phosphonate derived from glyphosate degradation, was evaluated. The best degradation performance was observed for bacteria from the genera Achromobacter, Agrobacterium and Ochrobactrum. The genome of the highly efficient GBH degrader Agrobacterium tumefaciens CHLDO was sequenced revealing the presence of a phn cluster, responsible for phosphonate metabolization. Expression analysis of A. tumefaciens CHLDO phn genes in the presence of 1.5 mM GBH compared to inorganic phosphorous showed that most of them are highly expressed during growth in the presence of the herbicide, suggesting a strong participation of phn cluster in GBH degradation. The importance of discovering new bacterial strains and the value of deciphering molecular determinants of GBH degradation give promising tools for bioremediation techniques to be used in glyphosate-contaminated environments is discussed.

Participation of two general stress response proteins from Xanthomonas citri subsp. citri in environmental stress adaptation and virulence.

Xanthomonas citri subsp. citri (Xcc) is the bacteria responsible for citrus canker. During its life cycle Xcc is found on leaves as epiphyte, where desiccation conditions may occur. In this work, two Xcc genes, XAC0100 and XAC4007, predicted in silico to be involved in general stress response, were studied under salt, osmotic, desiccation, oxidative and freezing stress, and during plant-pathogen interaction. Expression of XAC0100 and XAC4007 genes was induced under these stress conditions. Disruption of both genes in Xcc caused decreased bacterial culturability under desiccation, freezing, osmotic and oxidative stress. Importantly, the lack of these genes impaired Xcc epiphytic fitness. Both Xac0100 and Xac4007 recombinant proteins showed protective effects on Xanthomonas cells subjected to drought stress. Also, Escherichia coli overexpressing Xac4007 showed a better performance under standard culture, saline and osmotic stress and were more tolerant to freezing and oxidative stress than wild type E. coli. Moreover, both Xac0100 and Xac4007 recombinant proteins were able to prevent the freeze-thaw-induced inactivation of L-Lactate dehydrogenase. In conclusion, Xac0100 and Xac4007 have a relevant role as bacteria and protein protectors; and these proteins are crucial to bacterial pathogens that must face environmental stressful conditions that compromise the accomplishment of the complete virulence process.

A novel Xanthomonas citri subsp. citri NADPH quinone reductase involved in salt stress response and virulence.

BACKGROUND: Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker is maintained as an epiphyte on citrus leaves until entering the plant tissue. During epiphytic survival, bacteria may encounter low water availability that challenges the infection process. Proteomics analyses of Xcc under saline stress, mimicking the conditions found during epiphytic survival, showed increased abundance of a putative NAD(P)H dehydrogenase encoded by XAC2229. METHODS: Expression levels of XAC2229 and a Xcc mutant in XAC2229 were analyzed in salt and oxidative stress and during plant-pathogen interaction. An Escherichia coli expressing XAC2229 was obtained, and the role of this protein in oxidative stress resistance and in reactive oxygen species production was studied. Finally, Xac2229 protein was purified, spectrophotometric and cofactor analyses were done and enzymatic activities determined. RESULTS: XAC2229 was expressed under salt stress and during plant-pathogen interaction. ΔXAC2229 mutant showed less number of cankers and impaired epiphytic survival than the wild type strain. ΔXAC2229 survived less in the presence of H2O2 and produced more reactive oxygen species and thiobarbituric acid-reactive substances than the wild type strain. Similar results were observed for E. coli expressing XAC2229. Xac2229 is a FAD containing flavoprotein, displays diaphorase activity with an optimum at pH 6.0 and has quinone reductase activity using NADPH as an electron donor. CONCLUSIONS: A FAD containing flavoprotein from Xcc is a new NADPH quinone reductase required for bacterial virulence, particularly in Xcc epiphytic survival on citrus leaves. GENERAL SIGNIFICANCE: A novel protein involved in the worldwide disease citrus canker was characterized.

Proteome changes and physiological adaptations of the phytopathogen Xanthomonas citri subsp. citri under salt stress and their implications for virulence.

The bacterium Xanthomonas citri subsp. citri (Xcc) is responsible for the widely distributed disease citrus canker. In the last years, Xcc has become a model for the study of plant pathogens, and here we used this bacterium to examine stress on the pathogen during adaptions required for leaf colonization. In the first stages of citrus canker cycle, bacteria encounter low water availability and osmotic stress that can affect their maintenance on plant surfaces. To examine such conditions, we conducted a proteome analysis of Xcc grown in culture medium supplemented with 0.25 M sodium chloride and compared it to control conditions. We found that salt stress induced changes in known stress-induced proteins and also revealed novel stress response proteins. Moreover, some of the bacterial processes associated with bacterial fitness and virulence were modified under salt stress conditions. In particular, swimming, twitching and surface motilities were decreased, while biofilm formation was increased under salt stress. Other adaptations to high salt included reduced bacterial size and increased survival of bacteria exposed to oxidative stress. Furthermore, expression of type III protein secretion system related genes were augmented under salt stress condition. Our results offer new insight into molecular mechanisms that govern phytopathogen adaptation to harsh environments. These adaptations affect life cycle progression which in turn influences virulence.

Environmental Bacteria Involved in Manganese(II) Oxidation and Removal From Groundwater.

The presence of iron (Fe) and manganese (Mn) in groundwater is an important concern in populations that use it as source of drinking water. The ingestion of high concentrations of these metals may affect human health. In addition, these metals cause aesthetic and organoleptic problems that affect water quality and also induce corrosion in distribution networks, generating operational and system maintenance problems. Biological sand filter systems are widely used to remove Fe and Mn from groundwater since they are a cost-effective technology and minimize the use of chemical oxidants. In this work, the bacterial communities of two biological water treatment plants from Argentina, exposed to long term presence of Mn(II) and with a high Mn(II) removal efficiency, were characterized using 16S rRNA gene Illumina sequencing. Several selective media were used to culture Mn-oxidizing bacteria (MOB) and a large number of known MOB and several isolates that have never been reported before as MOB were cultivated. These bacteria were characterized to select those with the highest Mn(II) oxidation and biofilm formation capacities and also those that can oxidize Mn(II) at different environmental growth conditions. In addition, studies were performed to determine if the selected MOB were able to oxidize Mn(II) present in groundwater while immobilized on sand. This work allowed the isolation of several bacterial strains adequate to develop an inoculum applicable to improve Mn(II) removal efficiency of sand filter water treatment plants.

DOTAP, a lipidic transfection reagent, triggers Arabidopsis plant defense responses.

MAIN CONCLUSION: DOTAP triggers Arabidopsis thaliana immunity and by priming the defense response is able to reduce bacterial pathogen attack. DOTAP is a cationic lipid widely used as a liposomal transfection reagent and it has recently been identified as a strong activator of the innate immune system in animal cells. Plants are sessile organisms and unlike mammals, that have innate and acquired immunity, plants possess only innate immunity. A key feature of plant immunity is the ability to sense potentially dangerous signals, as it is the case for microbe-associated, pathogen-associated or damage-associated molecular patterns and by doing so, trigger an active defense response to cope with the perturbing stimulus. Here, we evaluated the effect of DOTAP in plant basal innate immunity. An initial plant defense response was induced by the cationic lipid DOTAP in the model plant Arabidopsis thaliana, assessed by callose deposition, reactive oxygen species production, and plant cell death. In addition, a proteomic analysis revealed that these responses are mirrored by changes in the plant proteome, such as up-regulation of proteins related to defense responses, including proteins involved in photorespiration, cysteine and oxylipin synthesis, and oxidative stress response; and down-regulation of enzymes related to photosynthesis. Furthermore, DOTAP was able to prime the defense response for later pathogenic challenges as in the case of the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Disease outcome was diminished in DOTAP-pre-treated leaves and bacterial growth was reduced 100 times compared to mock leaves. Therefore, DOTAP may be considered a good candidate as an elicitor for the study of plant immunity.

HrpE, the major component of the Xanthomonas type three protein secretion pilus, elicits plant immunity responses.

Like several pathogenic bacteria, Xanthomonas infect host plants through the secretion of effector proteins by the Hrp pilus of the Type Three Protein Secretion System (T3SS). HrpE protein was identified as the major structural component of this pilus. Here, using the Xanthomonas citri subsp. citri (Xcc) HrpE as a model, a novel role for this protein as an elicitor of plant defense responses was found. HrpE triggers defense responses in host and non-host plants revealed by the development of plant lesions, callose deposition, hydrogen peroxide production and increase in the expression levels of genes related to plant defense responses. Moreover, pre-infiltration of citrus or tomato leaves with HrpE impairs later Xanthomonas infections. Particularly, HrpE C-terminal region, conserved among Xanthomonas species, was sufficient to elicit these responses. HrpE was able to interact with plant Glycine-Rich Proteins from citrus (CsGRP) and Arabidopsis (AtGRP-3). Moreover, an Arabidopsis atgrp-3 knockout mutant lost the capacity to respond to HrpE. This work demonstrate that plants can recognize the conserved C-terminal region of the T3SS pilus HrpE protein as a danger signal to defend themselves against Xanthomonas, triggering defense responses that may be mediated by GRPs.

3-methylcrotonyl Coenzyme A (CoA) carboxylase complex is involved in the Xanthomonas citri subsp. citri lifestyle during citrus infection.

Citrus canker is a disease caused by the phytopathogen Xanthomonas citri subsp. citri (Xcc), bacterium which is unable to survive out of the host for extended periods of time. Once established inside the plant, the pathogen must compete for resources and evade the defenses of the host cell. However, a number of aspects of Xcc metabolic and nutritional state, during the epiphytic stage and at different phases of infection, are poorly characterized. The 3-methylcrotonyl-CoA carboxylase complex (MCC) is an essential enzyme for the catabolism of the branched-chain amino acid leucine, which prevents the accumulation of toxic intermediaries, facilitates the generation of branched chain fatty acids and/or provides energy to the cell. The MCC complexes belong to a group of acyl-CoA carboxylases (ACCase) enzymes dependent of biotin. In this work, we have identified two ORFs (XAC0263 and XAC0264) encoding for the α and ß subunits of an acyl-CoA carboxylase complex from Xanthomonas and demonstrated that this enzyme has MCC activity both in vitro and in vivo. We also found that this MCC complex is conserved in a group of pathogenic gram negative bacteria. The generation and analysis of an Xcc mutant strain deficient in MCC showed less canker lesions in the interaction with the host plant, suggesting that the expression of these proteins is necessary for Xcc fitness during infection.

Bacterial and plant natriuretic peptides improve plant defence responses against pathogens.

Plant natriuretic peptides (PNPs) have been implicated in the regulation of ions and water homeostasis, and their participation in the plant immune response has also been proposed. Xanthomonas citri ssp. citri contains a gene encoding a PNP-like protein (XacPNP) which has no homologues in other bacteria. XacPNP mimics its Arabidopsis thaliana homologue AtPNP-A by modifying host responses to create favourable conditions for pathogen survival. However, the ability of XacPNP to induce plant defence responses has not been investigated. In order to study further the role of XacPNP in vivo, A. thaliana lines over-expressing XacPNP, lines over-expressing AtPNP-A and AtPNP-A-deficient plants were generated. Plants over-expressing XacPNP or AtPNP-A showed larger stomatal aperture and were more resistant to saline or oxidative stress than were PNP-deficient lines. In order to study further the role of PNP in biotic stress responses, A. thaliana leaves were infiltrated with pure recombinant XacPNP, and showed enhanced expression of genes related to the defence response and a higher resistance to pathogen infections. Moreover, AtPNP-A expression increased in A. thaliana on Pseudomonas syringae pv. tomato (Pst) infection. This evidence led us to analyse the responses of the transgenic plants to pathogens. Plants over-expressing XacPNP or AtPNP-A were more resistant to Pst infection than control plants, whereas PNP-deficient plants were more susceptible and showed a stronger hypersensitive response when challenged with non-host bacteria. Therefore, XacPNP, acquired by horizontal gene transfer, is able to mimic PNP functions, even with an increase in plant defence responses.

Xanthomonas citri ssp. citri requires the outer membrane porin OprB for maximal virulence and biofilm formation.

Xanthomonas citri ssp. citri (Xcc) causes canker disease in citrus, and biofilm formation is critical for the disease cycle. OprB (Outer membrane protein B) has been shown previously to be more abundant in Xcc biofilms compared with the planktonic state. In this work, we showed that the loss of OprB in an oprB mutant abolishes bacterial biofilm formation and adherence to the host, and also compromises virulence and efficient epiphytic survival of the bacteria. Moreover, the oprB mutant is impaired in bacterial stress resistance. OprB belongs to a family of carbohydrate transport proteins, and the uptake of glucose is decreased in the mutant strain, indicating that OprB transports glucose. Loss of OprB leads to increased production of xanthan exopolysaccharide, and the carbohydrate intermediates of xanthan biosynthesis are also elevated in the mutant. The xanthan produced by the mutant has a higher viscosity and, unlike wild-type xanthan, completely lacks pyruvylation. Overall, these results suggest that Xcc reprogrammes its carbon metabolism when it senses a shortage of glucose input. The participation of OprB in the process of biofilm formation and virulence, as well as in metabolic changes to redirect the carbon flux, is discussed. Our results demonstrate the importance of environmental nutrient supply and glucose uptake via OprB for Xcc virulence.

XacFhaB adhesin, an important Xanthomonas citri ssp. citri virulence factor, is recognized as a pathogen-associated molecular pattern.

Adhesion to host tissue is one of the key steps of the bacterial pathogenic process. Xanthomonas citri ssp. citri possesses a non-fimbrial adhesin protein, XacFhaB, required for bacterial attachment, which we have previously demonstrated to be an important virulence factor for the development of citrus canker. XacFhaB is a 4753-residue-long protein with a predicted ß-helical fold structure, involved in bacterial aggregation, biofilm formation and adhesion to the host. In this work, to further characterize this protein and considering its large size, XacFhaB was dissected into three regions based on bioinformatic and structural analyses for functional studies. First, the capacity of these protein regions to aggregate bacterial cells was analysed. Two of these regions were able to form bacterial aggregates, with the most amino-terminal region being dispensable for this activity. Moreover, XacFhaB shows features resembling pathogen-associated molecular patterns (PAMPs), which are recognized by plants. As PAMPs activate plant basal immune responses, the role of the three XacFhaB regions as elicitors of these responses was investigated. All adhesin regions were able to induce basal immune responses in host and non-host plants, with a stronger activation by the carboxyl-terminal region. Furthermore, pre-infiltration of citrus leaves with XacFhaB regions impaired X. citri ssp. citri growth, confirming the induction of defence responses and restraint of citrus canker. This work reveals that adhesins from plant pathogens trigger plant defence responses, opening up new pathways for the development of protective strategies for disease control.

An Overview of Proteomics Tools for Understanding Plant Defense Against Pathogens.

Plant diseases are responsible for important losses in crops and cause serious impacts in agricultural production. In the last years, proteomics has been used to examine plant defense responses against pathogens. Such studies may be pioneer in the generation of crops with enhanced resistance. In this review, we focus on proteomics advances in the understanding of host and non-host resistance against pathogens.

The Amino Acid Arginine 210 of the Response Regulator HrpG of Xanthomonas citri subsp. citri Is Required for HrpG Function in Virulence.

Xanthomonas citri subsp. citri colonizes its hosts through the trafficking of effector proteins to the plant cell by the type III protein secretion system. In X. citri subsp. citri, as in other plant pathogens, the hrp cluster encodes the type III protein secretion system and is regulated by the transcription factors HrpG and HrpX. HrpG belongs to the OmpR family's response regulator of EnvZ/OmpR two-component signal transduction system. Here, we show that the arginine 210 residue is crucial for the transcriptional activity of HrpG revealed by the absence of disease in host plants and hypersensitive response in non-host plants when a strain carrying this point mutation is used in plant infiltration assays. Also, this strain showed decreased expression levels of hrp genes in bacteria grown in culture or when they were recovered from citrus leaves. Moreover, we show for the first time that HrpG binds to both hrpX and its own promoter, and the change of the arginine 210 by a cysteine does not prevent the binding to both promoters. Nevertheless, in vitro hrpX transcription was observed only with HrpG whereas no transcription was detected with the R210C mutant. HrpG was able to interact with itself as well as with the mutant R210C suggesting that it functions as a dimer. The mutant protein R210C showed altered protease sensitivity, suggesting that Arg210 is essential for protein active conformation and thus for transcriptional activity. Our results indicate that arginine 210 in HrpG, as it may occur with this conserved residue in other members of this family of response regulators, is not required for DNA binding whereas is essential for hrp genes transcription and therefore for pathogenicity and HR induction.

The dual nature of trehalose in citrus canker disease: a virulence factor for Xanthomonas citri subsp. citri and a trigger for plant defence responses.

Xanthomonas citri subsp. citri (Xcc) is a bacterial pathogen that causes citrus canker in susceptible Citrus spp. The Xcc genome contains genes encoding enzymes from three separate pathways of trehalose biosynthesis. Expression of genes encoding trehalose-6-phosphate synthase (otsA) and trehalose phosphatase (otsB) was highly induced during canker development, suggesting that the two-step pathway of trehalose biosynthesis via trehalose-6-phosphate has a function in pathogenesis. This pathway was eliminated from the bacterium by deletion of the otsA gene. The resulting XccΔotsA mutant produced less trehalose than the wild-type strain, was less resistant to salt and oxidative stresses, and was less able to colonize plant tissues. Gene expression and proteomic analyses of infected leaves showed that infection with XccΔotsA triggered only weak defence responses in the plant compared with infection with Xcc, and had less impact on the host plant's metabolism than the wild-type strain. These results suggested that trehalose of bacterial origin, synthesized via the otsA-otsB pathway, in Xcc, plays a role in modifying the host plant's metabolism to its own advantage but is also perceived by the plant as a sign of pathogen attack. Thus, trehalose biosynthesis has both positive and negative consequences for Xcc. On the one hand, it enables this bacterial pathogen to survive in the inhospitable environment of the leaf surface before infection and exploit the host plant's resources after infection, but on the other hand, it is a tell-tale sign of the pathogen's presence that triggers the plant to defend itself against infection.

The type III protein secretion system contributes to Xanthomonas citri subsp. citri biofilm formation.

BACKGROUND: Several bacterial plant pathogens colonize their hosts through the secretion of effector proteins by a Type III protein secretion system (T3SS). The role of T3SS in bacterial pathogenesis is well established but whether this system is involved in multicellular processes, such as bacterial biofilm formation has not been elucidated. Here, the phytopathogen Xanthomonas citri subsp. citri (X. citri) was used as a model to gain further insights about the role of the T3SS in biofilm formation. RESULTS: The capacity of biofilm formation of different X. citri T3SS mutants was compared to the wild type strain and it was observed that this secretion system was necessary for this process. Moreover, the T3SS mutants adhered proficiently to leaf surfaces but were impaired in leaf-associated growth. A proteomic study of biofilm cells showed that the lack of the T3SS causes changes in the expression of proteins involved in metabolic processes, energy generation, exopolysaccharide (EPS) production and bacterial motility as well as outer membrane proteins. Furthermore, EPS production and bacterial motility were also altered in the T3SS mutants. CONCLUSIONS: Our results indicate a novel role for T3SS in X. citri in the modulation of biofilm formation. Since this process increases X. citri virulence, this study reveals new functions of T3SS in pathogenesis.

Insights into xanthomonas axonopodis pv. citri biofilm through proteomics.

BACKGROUND: Xanthomonas axonopodis pv. citri (X. a. pv. citri) causes citrus canker that can result in defoliation and premature fruit drop with significant production losses worldwide. Biofilm formation is an important process in bacterial pathogens and several lines of evidence suggest that in X. a. pv. citri this process is a requirement to achieve maximal virulence since it has a major role in host interactions. In this study, proteomics was used to gain further insights into the functions of biofilms. RESULTS: In order to identify differentially expressed proteins, a comparative proteomic study using 2D difference gel electrophoresis was carried out on X. a. pv. citri mature biofilm and planktonic cells. The biofilm proteome showed major variations in the composition of outer membrane proteins and receptor or transport proteins. Among them, several porins and TonB-dependent receptor were differentially regulated in the biofilm compared to the planktonic cells, indicating that these proteins may serve in maintaining specific membrane-associated functions including signaling and cellular homeostasis. In biofilms, UDP-glucose dehydrogenase with a major role in exopolysaccharide production and the non-fimbrial adhesin YapH involved in adherence were over-expressed, while a polynucleotide phosphorylase that was demonstrated to negatively control biofilm formation in E. coli was down-regulated. In addition, several proteins involved in protein synthesis, folding and stabilization were up-regulated in biofilms. Interestingly, some proteins related to energy production, such as ATP-synthase were down-regulated in biofilms. Moreover, a number of enzymes of the tricarboxylic acid cycle were differentially expressed. In addition, X. a. pv. citri biofilms also showed down-regulation of several antioxidant enzymes. The respective gene expression patterns of several identified proteins in both X. a. pv. citri mature biofilm and planktonic cells were evaluated by quantitative real-time PCR and shown to consistently correlate with those deduced from the proteomic study. CONCLUSIONS: Differentially expressed proteins are enriched in functional categories. Firstly, proteins that are down-regulated in X. a. pv. citri biofilms are enriched for the gene ontology (GO) terms 'generation of precursor metabolites and energy' and secondly, the biofilm proteome mainly changes in 'outer membrane and receptor or transport'. We argue that the differentially expressed proteins have a critical role in maintaining a functional external structure as well as enabling appropriate flow of nutrients and signals specific to the biofilm lifestyle.