Progress 01/01/10 to 09/30/14
Outputs
Target Audience: The immediate target audience is the agricultural research community in both the public and private research institutions. Ultimately, the farmer will benefit when this knowledge is used in further research to develop disease management strategies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? A total of 10 graduate students were trained. Two of the trained students graduated with PhDs, 3 graduated with MS and 4 are still working on their research. Also, 4 high school students received training from the PI under Tennessee State University's Undergraduate Summer Research Internships in the College of Agriculture, Human and Natural Sciences. How have the results been disseminated to communities of interest? The findings from this project were presented to a wide audience for multiple years at 1. University-Wide Research Symposium at Tennessee State University 2. Annual Meeting of the American Society for Microbiology 3. Annual meeting of the American Phytopathological Society. What do you plan to do during the next reporting period to accomplish the goals? This is the final report. The project has ended.
Impacts
What was accomplished under these goals?
Pectobacterium carotovorum strain KD100 (lacZ mutant, Nalidixic Acid resistant) was mutagenized with mini-Tn5 lacZ1 carrying kanamycin (Km) resistance using biparental mating with E. coli S17-1. The transposon vector create transcriptional LacZ fusion in the truncated genes if the inserted in the direction of the gene flow. Colonies were selected on different media containing Km and Nal. To isolate mutants in host extract (CE)-induced genes, a total of about 31,122 mutants were screened for high expression of Beta-galactosidase in the presence of celery extract (CE). High LacZ expressing mutants were counter-selected on media without CE and Low expressing mutants were isolated. After repeated screening and evaluation of promoter activity of the mutants based on betagalactosidase (β-gal) assays, 98 mutants were obtained of which 83 were induced (> 5-fold) and 15 were repressed (<0.5-fold) in the presence of CE. These numbers were further reduced to 26 and 3, respectively. Semiquantitative assays on the selected (26 induced and 3 repressed) mutants showed increased production of cellulase (Cel), pectate lyase (Pel) and protease (Prt) in the presence of CE. Quantitative assays revealed that, compared to the parent, 6 of these mutants overproduced (> 2-fold) Pel while one was deficient (< 0.5-fold). Five mutants overproduced (≥1.4) Prt while 2 were deficient (< 0.5-fold). Compared to the parent, four mutants macerated at least 2-fold more potato tubers, and 6 mutants had reduced ability to macerate potato tubers. The process of identifying the genes into which the transposon inserted in these mutants is on-going as we have more mutants to determine their truncated, genes. Beside CE-regulated genes, the mutants were screened for altered production of extracellular enzymes in vitro. Several mutants were selected that either overproduced or were deficient in extracellular protease, one of the virulence factors produced by the bacterium to degrade the host cell wall. One of these mutants was completely deficient in Prt on nutrient-gelatin agar medium and was reduced in extracellular pectate lyase, polygalacturonase and cellulase activities and was less virulent in both celery petioles and carrot disks. The truncated gene was identified to be corA (cobalt resistance), that encodes a membrane protein responsible for the major transport of magnesium, nickel and cobalt into the cell. Although CorA mutants had the characteristic cobalt resistant phenotype as their closely related E. coli and Salmonella relatives, they maintained normal levels of intracellular magnesium. Through this project, mutants of Pectobacterium have been isolated. The affected genes in some of these mutants were identified and their role in disease development is being understood. We have implicated the major magnesium transporter, CorA in the pathogenesis of this soft rot erwinia. With more understanding, this information has the potential impact of being used in the design of a better management strategy for the diseases. The findings are contributing to our understanding of which bacterial genes play a role in disease development. Optimal concentrations of the host extract determined for the induction of enzyme production and β-galactosidase activities were 30% PE or CE and 0.2% polygalacturonic acid (PGA) supplemented into minimal medium (MM). Measurement of enzymatic activities of pectate lyase (Pel) from the supernatants of host-extract supplemented culture showed significant increases of 17- to-54-fold over MM in Ecc71 and 13- to 58-fold over MM in SCRI1043. The inducer(s) in extracts from the different hosts induce Pel activities to different degrees in the two strains. Taking together the responses of both P. carotovora Ecc71 and P. atrosepticum SCRI1043, the extracts with high induction were from celery petioles (106-fold), carrot roots (103-fold), potato tuber (92-fold) and turnip roots (81-fold). On the other end of the spectrum, the extract from onion bulb induced Pel to the lowest (31-fold) with the other host falling between. In the wild type Ecc71, the addition of each of the three extracts, PGA, CE or PE alone to the basal medium induced Pel activity by 13-, 40- and 102-fold respectively. There was even a higher induction when PGA+CE (84-fold) or PGA+PE (117-fold) were added to the basal medium. Thus plant extract and PGA had a synergistic effect on Pel activity in Ecc71 suggesting they have different constituents. Similarly, in the KdgR- mutant, induction of Pel production by the addition of CE or PE to the basal medium was approximately 19-fold. Unlike the parent, Pel production was not induced when the mutant was grown in MM+PGA or when PGA was added to CE or PE. Pel activities by the KdgR- mutant were higher than in the wild type strain in all the media except MM+PGA. Thus, in the KdgR- mutant, PGA does not induce Pel production. Moreover, the absence of any apparent increase in Pel production by the mutant when PGA was added to the extracts strongly suggests that the inducer(s) in the extracts (not PGA and its degradative intermediates) are responsible for the Pel induction in the mutant. Pectate lyase production by Ecc71 grown in autoclaved extracts from CE or PE was significantly higher than those from unautoclaved extracts at 30% extract concentration. The original inducing activity of the extract was also fully retained after freezing the extract at -80 oC for up to 6 months. Thus, the inducing molecule(s) was destroyed by neither heating nor freezing and could be described as thermostable and freeze-tolerant. The induction properties of the extracts were significantly (p≤0.05) reduced by pH-shock treatments. The negative effect of pH shock was more pronounced in PE that in CE. In both low and high pH-shock treatments and for both celery and potato extracts however, enzyme production levels remained significantly higher than in MM. The study on the effect of pH gradient and it showed that Pel induction by the extracts was pH-dependent. In MM+CE medium, induction of Pel in P. carotovorum strain KD100 dropped from 7.1-fold at pH 7.4 to 0.6-fold at pH 6.0 and to 0.6-fold at pH 9.0. The same trend was observed in MM+PE. These results suggest that inducer molecule(s) could only activate exoenzyme production under optimal pH.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Prestwich, P. & Dumenyo, C. K. 2014. Transformation of plasmids into Erwinia tracheiphila, the causal agent of bacterial wilt of cucurbits. 2014 TSU Research symposium
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Dumenyo, K,. Kersey, C. M., Hageman, B. 2014. Inactivation of rsmD of the soft rotting Pectobacterium atrosepticum leads to overproduction of exoenzymes and hypervirulence. Phytopathology 104(Suppl. 3):S3.35
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Kabir, M. N. & Dumenyo, C. K. 2014. Purification of the chemical inducer of soft rot disease from the extracts of celery (Apium graveolens) petioles. 2014 Tennessee State University Research symposium.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Adhikari, U & C. Korsi Dumenyo, 2014. Characterization of hypervirulent transposon Tn5 insertion RsmK- mutant of Pectobacterium carotovorum that also overproduces extracellular virulence factors. 2014 Tennessee State University Research symposium.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Scientific Research community Graduate and undergraduate students from underrepresented minority groups. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The following students were trained under this project resulting in their theses and dissertations for their MS and PhD degree, respectively. Paul Agyemang, PhD in Biological Sciences, May 2011. Project Title: Interaction between soft rot erwinia and host signals. Caleb Kersey, PhD in Biological Sciences, May 2011. Project title: Regulation of Pectobacterium virulence by membrane transporters. Sean Taylor, MS in Agricultural Sciences Dec 2012. Thesis Title: Genetic and physiological characterization of Erwinia tracheiphilla, the pathogen that causes bacterial wilt of cucumber. Roodie Johnson, MS in Agricultural Sciences, May 2013. Project Title: Host signal regulation of soft rot virulence. How have the results been disseminated to communities of interest? Publication: Kersey, C. M., Agyemang, P. A. and Dumenyo, C.K. 2012. CorA, the magnesium/nickel/cobalt transporter, affects virulence and extracellular enzyme production in the soft rot pathogen Pectobacterium carotovorum. Molecular Plant Pathology 13(1): 58-71 Conference Presentations Dumenyo, CK, Kersey, CM, Agyemang, PA, and Hageman, BH. 2011. Bacterial soft rot disease: Identification of the players involved in Pectobacterium-host interactions. Abstract # O-SP-S-5. ARD Symposium, Atlanta, GA. April 9-13, 2011. Kersey, CM and Dumenyo, CK. 2011. The Magnesium, Nickel, Cobalt transporter, CorA affects exoenzyme production and virulence in the soft rot pathogen, Pectobacterium carotovorum. Abstract # P-FS-G-11. ARD Symposium, Atlanta, GA. April 9-13, 2011. Kersey, C. & Dumenyo, C. K. 2010. CorA, a magnesium/nickel/cobalt transporter is required for full virulence in the soft rot pathogen, Pectobacterium carotovorum. Poster # 532-P. Annual Meeting of the American Phytopaythological Society, 7-11 August 2010, Charlotte, NC. Phytopathology 100:S61 Agyemang, P. and Dumenyo, C. K. 2010. Isolation and characterization of Pectobacterium carotovorum mutnats in host signal-responsive genes. Poster # 164-P. Annual Meeting of the American Phytopaythological Society, 7-11 August 2010, Charlotte, NC. Phytopathology 100:S3 Agyemang, P., Kersey, C. and Dumenyo, C. K. 2010. Initial characterization of plant metabolites inducing virulence in the soft rot pathogens, Pectobacterium spp. Poster # TP14, 32nd Annual University-wide research symposium. Tenn. State University. March 15-19, 2010 Kersey, C. and Dumenyo, C. K. 2010. Characterization of a Tn5 mutant of the soft rot bacterium, Pectobacterium carotovorum. Poster # TA8, 32nd Annual University-wide research symposium. Tenn. State University. March 15-19, 2010 Dumenyo, C. Korsi. 2009. Flow cytometric analysis of gene expression in the pathogenic bacterium, Erwinia carotovora subspecies atroseptica. Oral Presentation at the 31st Annual University Wide Research Symposium. TSU Dumenyo, C.K. Verbene-Sutton, S. and Ghazi, S. 2009. Flow cytometric analysis and sorting of host signal inducible transposon insertion mutants of Erwinia carotovora subsp. atroseptica. 15th Biennial ARD Research Symposium. Atlanta, GA. Dissertations/Theses 1. Kersey, Caleb M. 2011. Regulation of Pectobacterium virulence by membrane transporters. PhD Dissertation, Tennessee State University, Nashville, TN, USA. 2. Agyemang, Paul A. 2011. Interaction between soft rot erwinia and host signals. PhD. Dissertation. Tennessee State University, Nashville, TN, USA. 3. Johnson, Roodie 2013. Isolation of host extract induced mutants of Pectobacterium carotovorum by transposon mutagenesis. MS Thesis. Tennessee State University, Nashville, TN, USA What do you plan to do during the next reporting period to accomplish the goals? This is final Report
Impacts
What was accomplished under these goals?
Pectobacterium carotovorum strain KD100 (lacZ mutant, Nalidixic Acid resistant) was mutagenized with mini-Tn5 lacZ1 carrying kanamycin (Km) resistance using biparental mating with E. coli S17-1. The transposon vector creates transcriptional LacZ fusion in the truncated genes if it inserts in the direction of the gene flow. Colonies were selected on different media containing Km and Nal. To isolate mutants in host extract (CE)-induced genes, a total of about 31,122 mutants were screened for high expression of Beta-galactosidase in the presence of celery extract (CE). High LacZ expressing mutants were counter-selected on media without CE and low expressing mutants were isolated. After repeated screening and evaluation of promoter activity of the mutants based on betagalactosidase assays, 98 mutants were obtained of which 83 were induced (> 5-fold) and 15 were repressed (<0.5-fold) in the presence of CE. These numbers were further reduced to 26 and 3, respectively. Semiquantitative assays on the selected (26 induced and 3 repressed) mutants showed increased production of cellulase (Cel), pectate lyase (Pel) and protease (Prt) in the presence of CE. Quantitative assays revealed that, compared to the parent, 6 of these mutants overproduced (> 2-fold) pectate lyase while one was deficient (< 0.5-fold). Five mutants overproduced (≥1.4) Prt while 2 were deficient (< 0.5-fold). Compared to the parent, four mutants macerated at least 2-fold more potato than the parent, and 6 mutants had reduced ability to macerate potato tubers. The process of identifying the genes into which the transposon inserted in these mutants is on-going. Beside CE-regulated genes, the mutants were screened for altered production of extracellular enzymes in vitro. Several mutants were selected that either overproduced or were deficient in extracellular protease. One of these mutants was characterized further. The mutant was completely deficient in Prt on nutrient-gelatin agar medium and reduced in extracellular pectate lyase, polygalacturonase and cellulase activities and was less virulent in both celery petioles and carrot disks. The truncated gene was identified to be corA (cobalt resistance), that encodes a membrane protein responsible for the major transport of magnesium, nickel and cobalt into the cell. Although CorA mutants had the characteristic cobalt resistant phenotype as their E. coli and Salmonella relatives, they maintained normal levels of intracellular magnesium. We have determined the optimum concentration of host extract suitable for induction of extracellular enzyme production in the soft rot pathogens, Pectobacterium spp. Pectate lyase (Pel) activity was measured in P. carotovorum strain Ecc71 and in P. atrosepticum strain SCRI1043 grown in minimal medium supplemented with varying concentrations of juiced and filtered (GJF) or autoclaved (GJA) extract of celery petioles (CE) or potato tubers (PE). The enzymes, Pel and β-galactosidase activities of test tube-grown cultures were also assayed in Pectobacterium strain KD100 carrying the pel1-lacZ plasmid to determine the response of these bacteria to the extracts. We studied how widely distributed the inducer(s) might be among different host plant species of soft rot with GJF extracts from ten fruits or vegetables which also serve as hosts to soft rot disease. The extracts were supplemented in minimal medium (MM) and activities of Pel were measured from the culture supernatants. Since the chemical identity of the inducer in the host extracts is unknown, we determined if it is the same or different from polygalacturonic acid (PGA) by comparing the responses of a KdgR- mutant, (AC5073) and its parent, Ecc71 to host extracts. The two strains were grown in MM supplemented with PGA (0.2%, wt vol-1), GJF/CE or GJF/PE (30%, vol vol-1) in culture flasks. Following the determination that the inducer molecule in the host extracts is different from PGA, we determined some physical properties of the inducer and characterized the induction process. We also determined the heat-stability of the inducer molecule by heat-treating the extract before assaying for enzyme activities. The extracts and induction reactions were also pH-treated to determine the effects of pH on both. We have developed a procedure for the purification of the inducer from celery. Celery petioles are chopped up and air-dried and ground into powder. To determine the best solvent for the extraction of the inducer, we tried numerous organic solvents with a wide range of polarity. The solvent in which the extracted induced enzyme production the highest was methanol. Bioassay-guided extraction and purification of celery extract to purify and characterize the natural was our first step of identification and structural determination. Celery extract was dissolved in methanol and partially purified by dialysis through a 1000 Da membrane followed by hexane solvent extraction. Throughout the extraction and purification, the extract retained bioactivity of induction of Pel production comparable to extract obtained by grating and juicing celery petioles. The hexane extracted inducing fraction was dried and fractionated through CombiFlash chromatography. Chemical analysis of the resulting extract revealed the presence of several compounds in the extract which could be candidate inducers of Pel production. Bioassay guided separation and purification will continue and pure inducer will be analyzed by high performance liquid chromatography, NMR, IR and subsequent structural determination of the inducer(s) by analytical methods.
Publications
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: We have characterized another mutant from the pool of mutants isolated initially through transposon mutagenesis using mini-Tn lacZ1. This mutant, designated KD200, exhibited an increase in extracellular protease activity. The mutant KD200 was tested for pectolytic activity on polygalacturonate yeast extract agar (PYA) medium and showed a similar increase in enzymatic activities compared to the parent KD100. Increased enzymatic activity of mutant KD200 was further confirmed using semi-quantitative agar plate assays. The levels of pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt) were all notably higher in KD200 as compared to KD100. To accurately measure the increase in enzyme production in mutant KD200, parent KD100 and KD200 were grown in minimal medium and the culture supernatant was assayed for pectate lyase (Pel) activity. The production of Pel in KD200 was approximately 32 times higher than its parent KD100. This level of enzyme activity is one of the highest we have seen and is typically only found in mutants of strong negative regulators of exoenzyme such as rsmA, hexA, and rsmC. To determine how this high enzyme producing mutant would respond to inducers present in host extracts, KD200 and the parent KD100 were grown in celery extract (CE)-supplemented minimal medium (MM+CE) and the culture supernatant was measured for Pel activity. The mutant KD200 was only induced further about 4 fold in the presence of CE while the parent KD100 was induced 16 fold higher than MM. The increased activities of the major exoenzymes in mutant KD200 corresponded with an increase in transcripts of these enzymes, Pel, Peh, Cel, and Prt as measured by semi-quantitative RT-PCR. The differences in transcript levels observed using semi-quantitative RT-PCR assays were further quantified by measuring the expression with a real time RT-qPCR using SYBR Green detection system. Transcript levels of isozymes of pectale lyase, pel-1; polygalacturonase, peh-1; cellulose, celV; and protease, prtW were approximately 8, 24, 3 and 19 times higher in mutant KD200 respectively. These data confirm that the mutation in K200 produced an increase in exoenzyme transcripts and ultimately elevated enzyme activity. Primers were used to identify the gene truncated by insertion of the transposon in KD200 by sequencing across the transposon junction into the flanking genomic sequence. The generated DNA sequence was used as query to search against genomic databases of Pectobacterium and Dickeya spp. Based on the genomic sequence of P. carotovorum PC1 (NC_012917.1), the transposon inserted at base 41363. The transposon junction sequence revealed a 90% identity (with an E-value 1e-58.) with segments of P. carotovora subsp. carotovorum PC1 gene (PC1_0037) and P. carotovora SCRI1043 (ECA_4400). These genes are annotated as an anion transporter and putative sodium: sulfate symporter respectively, thus the gene was designated nssAEcc71 for Na SO4 symporter. PARTICIPANTS: Korsi Dumenyo, Caleb Kersey, Paul Agyemang TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Through this project, we have shown that: 1. The P. carotovorum NssA had increased production plant cell wall-degrading enzyme and was increased in tissue maceration in celery petioles. 2. Extrachromosomal copies of nssA+ did not complement the NssA mutant. Levels of nssA transcripts were increased in NssA mutant as compared to its parental strain 3. These findings implicate nssA in virulence and suggest KD200 overexpresses nssA.
Publications
- Kersey, C.M. and Dumenyo, C.K. 2012 Regulation of Expression of CorA, a Virulence Factor and Magnesium, Nickel, and Cobalt Transporter in the Soft Rot Pathogen, Pectobacterium Carotovorum, Phytopathology, 102 (2012), S4.32.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: We have determined the optimum concentration of host extract suitable for induction of extracellular enzyme production in the soft rot pathogens, Pectobacterium spp. Pectate lyase (Pel) activity was measured in P. carotovorum strain Ecc71 and in P. atrosepticum strain SCRI1043 grown in minimal medium supplemented with varying concentrations of juiced and filtered (GJF) or autoclaved (GJA) extract of celery petioles (CE) or potato tubers (PE). The enzymes, Pel and β-galactosidase activities of test tube-grown cultures were also assayed in Pectobacterium strain KD100 carrying the pel1-lacZ plasmid to determine the response of these bacteria to the extracts. We studied how widely distributed the inducer(s) might be among different host plant species of soft rot with GJF extracts from ten fruits or vegetables which also serve as hosts to soft rot disease. The extracts were supplemented in minimal medium (MM) and activities of Pel were measured from the culture supernatants. Since the chemical identity of the inducer in the host extracts is unknown, we determined if it is the same or different from polygalacturonic acid (PGA) by comparing the responses of a KdgR- mutant, (AC5073) and its parent, Ecc71 to host extracts. The two strains were grown in MM supplemented with PGA (0.2%, wt vol-1), GJF/CE or GJF/PE (30%, vol vol-1) in culture flasks. Following the determination that the inducer molecule in the host extracts is different from PGA, we determined some physical properties of the inducer and characterized the induction process. We also determined the heat-stability of the inducer molecule by heat-treating the extract before assaying for enzyme activities. The extracts and induction reactions were also pH-treated to determine the effects of pH on both. PARTICIPANTS: Korsi Dumenyo, PI; Paul Agyemang, Graduate Student; Caleb Kersey, Graduate Student TARGET AUDIENCES: Agricultural research, education and extension community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Optimal concentrations of the host extract determined for the induction of enzyme production and β-galactosidase activities were 30% PE or CE and 0.2% polygalacturonic acid (PGA) supplemented into minimal medium (MM). Measurement of enzymatic activities of pectate lyase (Pel) from the supernatants of host-extract supplemented culture showed significant increases of 17- to-54-fold over MM in Ecc71 and 13- to 58-fold over MM in SCRI1043. The inducer(s) in extracts from the different hosts induce Pel activities to different degrees in the two strains. Taking together the responses of both P. carotovora Ecc71 and P. atrosepticum SCRI1043, the extracts with high induction were from celery petioles (106-fold), carrot roots (103-fold), potato tuber (92-fold) and turnip roots (81-fold). On the other end of the spectrum, the extract from onion bulb induced Pel to the lowest (31-fold) with the other host falling between. In the wild type Ecc71, the addition of each of the three extracts, PGA, CE or PE alone to the basal medium induced Pel activity by 13-, 40- and 102-fold respectively. There was even a higher induction when PGA+CE (84-fold) or PGA+PE (117-fold) were added to the basal medium. Thus plant extract and PGA had a synergistic effect on Pel activity in Ecc71 suggesting they have different constituents. Similarly, in the KdgR- mutant, induction of Pel production by the addition of CE or PE to the basal medium was approximately 19-fold. Unlike the parent, Pel production was not induced when the mutant was grown in MM+PGA or when PGA was added to CE or PE. Pel activities by the KdgR- mutant were higher than in the wild type strain in all the media except MM+PGA. Thus, in the KdgR- mutant, PGA does not induce Pel production. Moreover, the absence of any apparent increase in Pel production by the mutant when PGA was added to the extracts strongly suggests that the inducer(s) in the extracts (not PGA and its degradative intermediates) are responsible for the Pel induction in the mutant. Pectate lyase production by Ecc71 grown in autoclaved extracts from CE or PE was significantly higher than those from unautoclaved extracts at 30% extract concentration. The original inducing activity of the extract was also fully retained after freezing the extract at -80oC for up to 6 months. Thus, the inducing molecule(s) was destroyed by neither heating nor freezing and could be described as thermostable and freeze-tolerant. The induction properties of the extracts were significantly (p≤0.05) reduced by pH-shock treatments. The negative effect of pH shock was more pronounced in PE that in CE. In both low and high pH-shock treatments and for both celery and potato extracts however, enzyme production levels remained significantly higher than in MM. The was a study on the effect of pH gradient and it showed that Pel induction by the extracts was pH-dependent. In MM+CE medium, induction of Pel in P. carotovorum strain KD100 dropped from 7.1-fold at pH 7.4 to 0.6-fold at pH 6.0 and to 0.6-fold at pH 9.0. The same trend was observed in MM+PE. These results suggest that inducer molecule(s) could only activate exoenzyme production under optimal pH.
Publications
- Kersey, C. M., Agyemang, P. A. and Dumenyo, C.K. 2012. CorA, the magnesium/nickel/cobalt transporter, affects virulence and extracellular enzyme production in the soft rot pathogen Pectobacterium carotovorum. Molecular Plant Pathology 13(1): 58-71.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Pectobacterium carotovorum strain KD100 (lacZ mutant, Nalidixic Acid resistant) was mutagenized with mini-Tn5 lacZ1 carrying kanamycin (Km) resistance using biparental mating with E. coli S17-1. The transposon vector creates transcriptional LacZ fusion in the truncated genes if it inserts in the direction of the gene flow. Colonies were selected on different media containing Km and Nal. To isolate mutants in host extract (CE)-induced genes, a total of about 31,122 mutants were screened for high expression of Beta-galactosidase in the presence of celery extract (CE). High LacZ expressing mutants were counter-selected on media without CE and low expressing mutants were isolated. After repeated screening and evaluation of promoter activity of the mutants based on betagalactosidase assays, 98 mutants were obtained of which 83 were induced (> 5-fold) and 15 were repressed (<0.5-fold) in the presence of CE. These numbers were further reduced to 26 and 3, respectively. Semiquantitative assays on the selected (26 induced and 3 repressed) mutants showed increased production of cellulase (Cel), pectate lyase (Pel) and protease (Prt) in the presence of CE. Quantitative assays revealed that, compared to the parent, 6 of these mutants overproduced (> 2-fold) pectate lyase while one was deficient (< 0.5-fold). Five mutants overproduced (≥1.4) Prt while 2 were deficient (< 0.5-fold). Compared to the parent, four mutants macerated at least 2-fold more potato than the parent, and 6 mutants had reduced ability to macerate potato tubers. The process of identifying the genes into which the transposon inserted in these mutants is on-going. Beside CE-regulated genes, the mutants were screened for altered production of extracellular enzymes in vitro. Several mutants were selected that either overproduced or were deficient in extracellular protease. One of these mutants was characterized further. The mutant was completely deficient in Prt on nutrient-gelatin agar medium and reduced in extracellular pectate lyase, polygalacturonase and cellulase activities and was less virulent in both celery petioles and carrot disks. The truncated gene was identified to be corA (cobalt resistance), that encodes a membrane protein responsible for the major transport of magnesium, nickel and cobalt into the cell. Although CorA mutants had the characteristic cobalt resistant phenotype as their E. coli and Salmonella relatives, they maintained normal levels of intracellular magnesium. PARTICIPANTS: Dumenyo, C. Korsi (PI) Kersey, C. M. (Graduate Student) Agyemang, P. A. (Graduate Student) Hageman, B. H. (Graduate Student) TARGET AUDIENCES: Agricultural Research Community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Through this project, mutants of Pectobacterium have been isolated. The affected genes in these mutants will be identified and their role in disease development will be understood. Thus far, we have implicated the major magnesium transporter, CorA in the pathogenesis of this soft rot erwinia. With more understanding, this information could be used in the design of a better management strategy for the diseases. The findings are contributing to the our understanding of which bacterial genes play a role in disease development.
Publications
- No publications reported this period
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