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.
Xanthomonas axonopodis pv. citri, the phytopathogen responsible for citrus canker has a number of protein secretion systems and among them, at least one type V protein secretion system belonging to ...the two-partner secretion pathway. This system is mainly associated to the translocation of large proteins such as adhesins to the outer membrane of several pathogens. Xanthomonas axonopodis pv. citri possess a filamentous hemagglutinin-like protein in close vicinity to its putative transporter protein, XacFhaB and XacFhaC, respectively. Expression analysis indicated that XacFhaB was induced in planta during plant-pathogen interaction. By mutation analysis of XacFhaB and XacFhaC genes we determined that XacFhaB is involved in virulence both in epiphytic and wound inoculations, displaying more dispersed and fewer canker lesions. Unexpectedly, the XacFhaC mutant in the transporter protein produced an intermediate virulence phenotype resembling wild type infection, suggesting that XacFhaB could be secreted by another partner different from XacFhaC. Moreover, XacFhaB mutants showed a general lack of adhesion and were affected in leaf surface attachment and biofilm formation. In agreement with the in planta phenotype, adhesin lacking cells moved faster in swarming plates. Since no hyperflagellation phenotype was observed in this bacteria, the faster movement may be attributed to the lack of cell-to-cell aggregation. Moreover, XacFhaB mutants secreted more exopolysaccharide that in turn may facilitate its motility. Our results suggest that this hemagglutinin-like protein is required for tissue colonization being mainly involved in surface attachment and biofilm formation, and that plant tissue attachment and cell-to-cell aggregation are dependent on the coordinated action of adhesin molecules and exopolysaccharides.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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
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
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.
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.
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 upregulation 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.
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.
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.
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.
A FAD containing flavoprotein from Xcc is a new NADPH quinone reductase required for bacterial virulence, particularly in Xcc epiphytic survival on citrus leaves.
A novel protein involved in the worldwide disease citrus canker was characterized.
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•Xanthomonas citri XAC2229 gene is required for bacterial epiphytic survival.•Xac2229 is a FAD-containing enzyme that preferentially uses NADPH as electron donor.•Xac2229 is a dehydrogenase with quinone reductase activity.
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 β 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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are ...important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta- or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo2Hex6GalA3Fuc3NAcRha4 and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response.
Background: Xanthomonas axonopodis pv. citri (Xac) is an obligate aerobic phytopathogen constantly exposed to hydrogen peroxide produced by normal aerobic respiration and by the plant defense ...response during plant-pathogen interactions. Four putative catalase genes have been identified in silico in the Xac genome, designated as katE, catB, srpA (monofunctional catalases) and katG (bifunctional catalase). Methodology/Principal Findings: Xac catalase activity was analyzed using native gel electrophoresis and semi-quantitative RT-PCR. We demonstrated that the catalase activity pattern was regulated in different growth stages displaying the highest levels during the stationary phase. KatE was the most active catalase in this phase of growth. At this stage cells were more resistant to hydrogen peroxide as was determined by the analysis of CFU after the exposition to different H2O2 concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H2O2 resistance after treatment with sub-lethal concentrations of the oxidant. In the plant-like medium XVM2 the expression of KatE was strongly induced and in this medium Xac was more resistant to H2O2. A XackatE mutant strain was constructed by insertional mutagenesis. We observed that catalase induction in stationary phase was lost meanwhile the adaptive response to peroxide was maintained in this mutant. Finally, the XackatE strain was assayed in planta during host plant interaction rendering a less aggressive phenotype with a minor canker formation. Conclusions: Our results confirmed that in contrast to other Xanthomonas species, Xac catalase-specific activity is induced during the stationary phase of growth in parallel with the bacterial resistance to peroxide challenge. Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides. The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process. Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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