Essential role of the czc determinant for cadmium, cobalt and zinc resistance in Gluconacetobacter diazotrophicus PAl 5

The mechanisms of cadmium, cobalt and zinc resistance were characterized in the plant-growth-promoting bacterium Gluconacetobacter diazotrophicus PAl 5. The resistance level of the wild-type strain was evaluated through the establishment of minimum inhibitory concentrations (MIC) of the soluble compounds CdCl2·H2O, CoCl2·6H2O and ZnCl2. Gluconacetobacter diazotrophicus PAl 5 was resistant to high concentrations of Cd, Co and Zn, with MICs of 1.2, 20 and 20 mM, respectively. Screening of an insertion library from transposon EZ-Tn5<R6Kyori/KAN-2> in the presence of ZnO revealed that the mutant GDP30H3 was unable to grow in the presence of the compound. This mutant was also highly sensitive to CdCl2·H2O, CoCl2·6H2O and ZnCl2. Molecular characterization established that the mutation affected the czcA gene, which encodes a protein involved in metal efflux. In silico analysis showed that czcA is a component of the czcCBARS operon together with four other genes. This work provides evidence of the high tolerance of G. diazotrophicus PAl 5 to heavy metalsand that czc is a determinant for metal resistance in this bacterium. [Int Microbiol 2012; 15(2):69-78

Incorporação de lodo da estação de tratamento de esgoto (ETE) em cerâmica vermelha

Resumo Este trabalho teve por objetivo avaliar a incorporação de resíduo de estação de tratamento de esgoto (ETE) na produção de cerâmica vermelha. As matérias-primas utilizadas foram: massa cerâmica argilosa e resíduo de ETE provenientes do município de Campos dos Goytacazes - RJ. As matérias-primas foram caracterizadas pelas técnicas de fluorescência de raios X e difração de raios X; além disso, foi determinada a área de superfície específica das mesmas por meio das técnicas de BET e azul de metileno. O comportamento térmico do resíduo de ETE foi avaliado por meio de análise termogravimétrica. Formulações foram preparadas com 0, 2,5, 10 e 15% em massa de resíduo de ETE incorporados à massa argilosa. Corpos de prova foram confeccionados por prensagem uniaxial a 20 MPa com 8% de umidade para calcinação a 950 °C. As propriedades físicas e mecânicas avaliadas das cerâmicas calcinadas foram retração linear, absorção de água e resistência à compressão. A microestrutura destas peças foi investigada com auxílio de microscopia eletrônica de varredura. Os resultados indicaram que este tipo de resíduo deve ser incorporado em pequenas quantidades (até 2,5%) para não prejudicar as propriedades físicas e mecânicas da cerâmica

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Summary. The mechanisms of cadmium, cobalt and zinc resistance were characterized in the plant-growth-promoting bacterium Gluconacetobacter diazotrophicus PAl 5. The resistance level of the wild-type strain was evaluated through the establishment of minimum inhibitory concentrations (MIC) of the soluble compounds CdCl 2 ·H 2 O, CoCl 2 ·6H 2 O and ZnCl 2 . Gluconacetobacter diazotrophicus PAl 5 was resistant to high concentrations of Cd, Co and Zn, with MICs of 1.2, 20 and 20 mM, respectively. Screening of an insertion library from transposon EZ-Tn5<R6Kyori/KAN-2> in the presence of ZnO revealed that the mutant GDP30H3 was unable to grow in the presence of the compound. This mutant was also highly sensitive to CdCl 2 ·H 2 O, CoCl 2 ·6H 2 O and ZnCl 2 . Molecular characterization established that the mutation affected the czcA gene, which encodes a protein involved in metal efflux. In silico analysis showed that czcA is a component of the czcCBARS operon together with four other genes. This work provides evidence of the high tolerance of G. diazotrophicus PAl 5 to heavy metals and that czc is a determinant for metal resistance in this bacterium. [Int Microbiol 2012; 15(2):69-78

Correction: Protein Poly(ADP-ribosyl)ation Regulates Arabidopsis Immune Gene Expression and Defense Responses.

[This corrects the article DOI: 10.1371/journal.pgen.1004936.]

Protein Poly(ADP-ribosyl)ation Regulates <i>Arabidopsis</i> Immune Gene Expression and Defense Responses

<div><p>Perception of microbe-associated molecular patterns (MAMPs) elicits transcriptional reprogramming in hosts and activates defense to pathogen attacks. The molecular mechanisms underlying plant pattern-triggered immunity remain elusive. A genetic screen identified <i>Arabidopsis</i> poly(ADP-ribose) glycohydrolase 1 (<i>atparg1</i>) mutant with elevated immune gene expression upon multiple MAMP and pathogen treatments. Poly(ADP-ribose) glycohydrolase (PARG) is predicted to remove poly(ADP-ribose) polymers on acceptor proteins modified by poly(ADP-ribose) polymerases (PARPs) with three PARPs and two PARGs in <i>Arabidopsis</i> genome. AtPARP1 and AtPARP2 possess poly(ADP-ribose) polymerase activity, and the activity of AtPARP2 was enhanced by MAMP treatment. AtPARG1, but not AtPARG2, carries glycohydrolase activity <i>in vivo</i> and <i>in vitro</i>. Importantly, mutation (G450R) in <i>atparg1</i> blocks its activity and the corresponding residue is highly conserved and essential for human HsPARG activity. Consistently, mutant <i>atparp1atparp2</i> plants exhibited compromised immune gene activation and enhanced susceptibility to pathogen infections. Our study indicates that protein poly(ADP-ribosyl)ation plays critical roles in plant immune gene expression and defense to pathogen attacks.</p></div

AtPARPs positively regulate <i>Arabidopsis</i> immunity.

<p>(<b>A</b>) The <i>atparp1/2</i> double mutant is more susceptible to <i>Psm</i> infection. WT (Col-0) and <i>atparp1/2</i> double mutant plants were hand-inoculated with <i>Psm</i> at OD₆₀₀  =  5 × 10⁻⁴, and the bacterial counting was performed 0 and 3 days post-inoculation (dpi). The data are shown as mean ± se from three independent repeats with Student's <i>t</i>-test. * indicates p<0.05 when compared to WT (Left panel). The disease symptom is shown at 3 dpi (right panel). (<b>B</b>) The <i>atparp1/2</i> double mutant is more susceptible to <i>Pst</i> DC3000Δ<i>avrPtoavrPtoB</i> infection. WT and <i>atparp1/2</i> double mutant plants were hand-inoculated with <i>Pst</i> DC3000<i>ΔavrPtoavrPtoB</i> at OD₆₀₀  =  5 × 10^-4, and the bacterial counting was performed 0 and 5 dpi (left panel) and the disease symptom is shown at 5 dpi (right panel). (<b>C</b>) Reduced immune gene expression in <i>atparp1/2</i> mutant. Ten-day-old seedlings were treated with 100 nM flg22 for 90 min for qRT-PCR analysis. The data are shown as means ± se from three biological repeats. * indicates p<0.05 when compared to WT. The above experiments were repeated 4 times with similar results.</p

The signature motif and residue G450 are essential for PARG enzymatic activity.

<p>(<b>A</b>) The conserved residues G264 and E273 in the PARG signature motif of AtPARG1 are required for its enzymatic activity (left part of top panel) and creating a conserved PARG signature motif in AtPARG2 did not make it enzymatically active (right part of top panel). The sequences of PARG signature motif in AtPARG1 and AtPARG2 are shown on the top with the polymorphic residue labeled in red. F457 locates outside of the PARG signature motif in AtPARG1 and is not required for its enzymatic activity. AtPARP2 proteins were auto-PARylated and further subjected for PARG assay with WT AtPARG1, AtPARG2 or different mutated variants of AtPARGs. The PARylated proteins were detected with an α-PAR Western blot (top panel) and protein inputs are shown with CBB staining (bottom panel). (<b>B</b>) G450 in AtPARG1 is an essential residue in human HsPARG. Alignment of sequences of AtPARG1, aggie2 and HsPARG around AtPARG1G450 (red) residue is shown on the top. The corresponding G867R mutation in HsPARG blocked its activity to hydrolyze auto-PARylated human HsPARP-1. The auto-PARylated human HsPARP-1 proteins were incubated with WT or mutant form of HsPARG for PARG assay. The PARylated proteins were detected with an α-PAR Western blot (top panel) and protein inputs are shown with CBB staining (middle & bottom panels). The above experiments were repeated 3 times with similar results.</p

AtPARG1, but not aggie2 or AtPARG2, has poly(ADP-ribose) glycohydrolase activity.

<p>(<b>A</b>) Schematic catalytic domain of AtPARG1, AtPARG2 and aggie2. The sequence of the PARG signature motif is shown and the number indicates the position of the first glycine (G) residue with a polymorphic residue G and L (leucine) between AtPARG1 and AtPARG2 highlighted in red. * denotes the point mutation in aggie2. (<b>B</b>) AtPARG1, but not AtPARG2 nor aggie2, possesses <i>in vitro</i> PAR glycohydrolase activity towards auto-PARylated AtPARP2 proteins detected by α-PAR antibody. MBP-AtPARP2 proteins were auto-PARylated and further subjected for <i>in vitro</i> PARG assay using GST-tagged AtPARG1, aggie2 or AtPARG2 proteins. The PARylated proteins were detected with an α-PAR Western blot (top panel) and the protein inputs are shown with CBB staining (bottom panel). (<b>C</b>) The AtPARG <i>in vitro</i> activity detected with ³²P-NAD⁺. AtPARG1, but not AtPARG2 or aggie2, possesses <i>in vitro</i> PARG activity towards auto-PARylated AtPARP2 proteins (left part of top panel) and AtPARG2 does not have PARG activity towards auto-PARylated AtPARP1 proteins (right part of top panel). MBP-AtPARP1 or AtPARP2 proteins were auto-PARylated and further subjected for <i>in vitro</i> PARG assays using GST-tagged PARG1, aggie2 or PARG2 proteins in the presence of ³²P-NAD⁺. The PARylated proteins were detected with autoradiograph (top panel) and the protein inputs are shown with CBB staining (bottom panel). (<b>D</b>) Protoplast-expressed AtPARG1 possesses PARG activity towards <i>in vitro</i> auto-PARylated AtPARP2 proteins. <i>Arabidopsis</i> protoplasts were transfected with <i>AtPARG1-FLAG</i> or vector control and treated with or without 100 nM flg22 for 15 min. PARG1 proteins were immunoprecipitated with α-FLAG antibody and subjected for <i>in vitro</i> PARG assay with <i>in vitro</i> auto-PARylated MBP-AtPARP2 proteins. The PARylated proteins were detected in an α-PAR Western blot (top panel), MBP-AtPARP2 protein input is shown with CBB staining (middle panel) and AtPARG1-FLAG protein expression in protoplasts is shown with an α-FLAG Western blot (bottom panel). (<b>E</b>) AtPARG1, but not AtPARG2, has <i>in vivo</i> PAR glycohydrolase activity. AtPARP2-FLAG was co-expressed with vector control, AtPARG1-HA or AtPARG2-HA in protoplasts and, the protoplasts were fed with ³²P-NAD⁺. The PARylated proteins were detected with autoradiograph after immunoprecipitation with α-FLAG antibody (top panel). The PARP and PARG protein expression was detected with Western blot (middle panels) and the protein loading is shown with Ponceau S staining (bottom panel). (<b>F</b>) Subcellular localization of AtPARG1 and AtPARG2 in protoplasts. AtPARG1-GFP or AtPARG2-GFP was transiently expressed in protoplasts and the images were taken 12 hr after transfection using a confocal microscope. NLS-RFP was co-transfected for nuclear localization control. The above experiments were repeated 3 times with similar results.</p

Elevated <i>pFRK1::LUC</i> expression and MAMP-triggered immune response in <i>aggie2</i> mutant.

<p>(<b>A</b>) Luciferase activity from 10-day-old <i>pFRK1::LUC</i> (WT) and <i>aggie2</i> seedlings treated with or without 10 nM flg22 for 12 hr. The photograph was taken with an EMCCD camera. The number below indicates quantified signal intensity shown as means ± se from 12 seedlings. (<b>B</b>) Time-course of <i>pFRK1::LUC</i> activity in response to 100 nM flg22 treatment. The data are shown as means ± se from at least 20 seedlings for each time point. (<b>C</b>) The <i>pFRK1::LUC</i> activity in response to different MAMPs. Ten-day-old seedlings were treated with 100 nM elf18, 50 µg/ml chitin, 1 µM LPS, or 500 ng/ml PGN for 12 hr. The data are shown as means ± se from at least 12 seedlings for each treatment. (<b>D</b>) The <i>pFRK1::LUC</i> activity triggered by different bacteria. Four-week-old soil-grown plants were hand-inoculated with different bacteria at the concentration of OD₆₀₀  =  0.5. The data are shown as means ± se from at least 12 leaves for each treatment at 24 hr post-inoculation (hpi). (<b>E</b>) flg22-induced callose deposition in <i>aggie2</i> mutant. Leaves of 6-week-old plants were infiltrated with 0.5 µM flg22 for 12 hr and callose deposits were detected by aniline blue staining and quantified by ImageJ software. (<b>F</b>) flg22-induced MAPK activation in <i>aggie2</i> mutant. Seedlings were treated with 100 nM flg22 and collected at the indicated time points. The MAPK activation was detected with an α-pErk antibody (top panel) and the protein loading was indicated by Ponceau S staining for RuBisCo (RBC) (bottom panel). (<b>G</b>) flg22-triggered ROS burst in <i>aggie2</i> mutant. Leave discs from 4-week-old plants were treated with H₂O or 100 nM flg22 over 30 min. The data are shown as means ± se from 20 leaf discs. (<b>H</b>) Endogenous MAMP-induced marker gene expression. Ten-day-old seedlings were treated with 100 nM flg22 for 30 and 90 min for qRT-PCR analysis. The data are shown as means ± se from three biological repeats with Student's <i>t</i>-test. * indicates p<0.05 and ** indicates p<0.01 when compared to WT. The above experiments were repeated 3 times with similar results.</p