Progress 01/09/15 to 09/30/17
Outputs
Target Audience:The target audience for this project is the agricultural, research, extension and education community in the US and across the world. This includes 1) students at all levels in agricultural sciences, 2) teachers at all levels of agricultural sciences, 3) agricultural researchers, and 4) agricultural extension personnel. Changes/Problems:Our biggest problem in this whole project was the technical challenges that we encountered in developing a protocol for constructing mutants in Erwinia tracheiphila. With E. tracheiphila being an enterobacteriaceae, we had the expectation that with little tweaking, we will be able to adopt protocols which had worked well with other organisms. This turned out not to be the case. However, because of the essentiality of mutant isolation protocol for the analysis of these pathogen leading to an effective bacterial wilt disease management strategy, attempts will continue at developing a working protocols for both random and site-directed mutagenesis in Erwinia tracheiphila. What opportunities for training and professional development has the project provided?The project provided for the training of four freshmen undergraduate students enrolled in the Biotechnology Concentration of BS-Agriculture program. One graduate student enrolled in MS-Agricultural Science worked in the project for two monts before the end of the project. The graduate student also has the opportunity to attend the annual meeting of Kentucky-Tennessee branch of the American Society for Microbiology where the PI gave a talk. Training opportunity was also given to an undergraduate student who worked in under the the PI's mentorship. How have the results been disseminated to communities of interest?Findings from this project have been disseminated through 1. Presentation in University-wide research symposium 2. Incoporation of the findings in the classes taught by the PI 3. Annual meeting of Tennessee/Kentucky branch of American Academy of sciences 4. Additional manuscripts are in preparation for submission. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We had modified the transposon vector, pCKD100 by digesting with the enzyme SphI and the linearized fragment was treated with the oligonucleotide adapter, 5' TACATG 3' to convert the site for the restriction enzyme, SphI into the overhanging ends of the enzyme NdeI. These ends are compatible with the ends of the DNA fragment containing the sacB gene. Following ligation of sacB fragment into the vector, the ligated product was transformed into E. coli strain, S17-1 lambda Pir that can replicate plasmids of R6K origin. Colonies were selected on Chloramphenicol-supplemented media. The 10% sucrose was to ensure the colonies did not grow, an indication that the sacB gene expressed, produced the enzyme levansucrase which killed the bacteria. The next confirmation test was to mate each of these clones into E. tracheiphila strain Hca1N. The clones were mated bi-parentally overnight into the recipient strain and the mating mixture was plated on medium containing nalidixic acid (Nal), chloramphenicol, 10% sucrose and with or without kanamycin. The sacB gene was to ensure that only clones which had lost the vector and sacB grow on sucrose while the Nal was to keep the donor strain from growing. The kanamycin was to select for transconjugants in which the transposon inserted downstream of an active promoter which then drives the expression of the promoter-less gfp-nptII gene cassette. Surprisingly, we did not obtain any transconjugants under this selection conditions with or without kanamycin. Since we did not obtain and colonies, we did not follow through with sequencing confirmation of the clones. We tried one last attempt to generate random mutants in E. tracheiphila using commercially available purified transposase. We have had many unsuccessful attempts with our own custom-designed transposon vector, pCKD100 that has additional features to help in analyzing the mutants. These features included carrying promoter-less gfp to allow for expression studies of the mutated gene, promoter-less nptII to allow for selection of mutants with insertions downstream of active promoters, R6K origin within the transposon for easy cloning and identification of the mutant genes and transfer of the mutation into other host backgrounds. For this, we tried using a simple transposon containing nptII gene flanked by the inverted repeat which serve as the recognition sites for the transposon. The transposon was PCR-amplified from miniTn5-Km and purified. The purified transposon and commercially acquired purified transposase enzyme were reconstituted into the transposome. Electrocompetent cells were prepared of E. tracheiphila strain Hca1 and the transposome was transformed into the competent cells. The electroporation mixture was incubated for two hours in shaker and plated on kanamycin-containing medium. The selected colonies are currently being tested for confirmation of transposition and mutation.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Dumenyo, C. K., 2017. Transposon-based strategies for genetic analysis in phytopathogenic Enterobacteriaceae. Presented at Fall 2017 meeting of Kentucky-Tennessee Branch of American Society for Microbiology. Nov 10-11, 2017. Cookeville, TN.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:The target audince for this project is the agricultural, research, extension and education community in the US and across the world. This includes 1) students at all levels in agricultural sciences, 2) teachers at all levels of agricultural sciences, 3) agricultural researchers, and 4) agricultural extension personnel. Changes/Problems:We encounted significant technical problems with the approach of purifying the transposase to be used to constiture the transposome in vitro for in vivo transpsome mutagenesis. Although we obtained a protein band on the gel that corresponded to the TnpA protein, functional assays of the protein did not yield any mutants. This lead us to try the approach of yet gain modifying the GFP transposon with sacB. These problems have led to a delay in the project time line. We expect to be back on track once the sacB modified transposon vectorsare functional. What opportunities for training and professional development has the project provided?The project provided for the training of one MS levelgraduate student who completed MS degree in December 2016. He wasemployed 50% as a graduate research assistant on this project while working on his MS degree in Agricultural Sciences. He had the opportunity to present his findings in local meeting:- University-wide research symposium, Annual meeting of Tennessee/Kentucky branch of American Society for Microbiology and the annual meeting of the Tennessee Academy of Science. Through the project, laboratory research experiential training was provided to three undergraduate students majoring in Biotechnology. These students worked with graduate students on various aspects of the project. One of these students was a high school intern in the lab the previous summer. The undergraduate students workin the laboratory as an undergraduate researcher under the Dean's Scholars Program. How have the results been disseminated to communities of interest?Findings from this project have been disseminated through 1. Publication in peer-reviewed journal 2. Presentation in University-wide research symposium 3. Invited lectures and talks on other campuses What do you plan to do during the next reporting period to accomplish the goals?We are going to evaluate the newly constructed modified transposon clones. Although only one of these is needed, all the obtained clones will be tested for thier ability to be transfereed and to transpose into the target genome. Because of the slow growth of Erwinia tracheiphila, the testing will be done inPectobacterium carotovorum, the faster-growing relative of E.tracheiphila. colonies will be selected on Chroramphenicol and nalidixic acid supplemented media with and without sucrose and/or Kanamycin. The cloned capable of transposition in Pectobacterium will be tested in E. tracheiphila. The clone that tests positive in these functional screens will be sequenced. Following confirmation by sequencing, one good clone will be used in the generation of transposon mutant library of Erwinia tracheiphila.Once established, the library of GFP fusion mutants will be screenedunder various conditions including different known inducers of virulence in other pathogenic bacterial systems.
Impacts
What was accomplished under these goals?
We had previously constructed, pCKD100, an R6K-based promoter-less gfp-nptII fusion Tn5 transposon vector for mutagenesis of E. tracheiphila. This vector was to be used in transposon mutagenesis of E. tracheiphila strains. However, we failed to obtain KmR or Gfp+ colonies following repeated electroporation of the plasmid into E. tracheiphila strains. Further investigations and observations led to the conclusion that E. tracheiphila strains could replicate this R6K-based plasmid. Therefore, it could not be used as a suicide vector in E. tracheiphila for random transposon mutagenesis. An alternate approached had to be considered. Production of Tn5 Transposase for composition of transposome We considered the option of in vivo mutagenesis using in vitro reconstituted transposase. We wanted to purity our own transposase and use it to reconstitute the transposome with the amplified transposon from pCKD100. We obtained pTXB1 (Addgene Plasmid #60240) a transposase over-expressing vector as a gift from Richard Sandberg. The plasmid was transformed into electrocompetent E. coli BL21(DE3). To purify the protein, one liter of culture was grown at 37°C to A600 of 0.9. The culture was then chilled, induced by IPTG and grown for additional 4 hours at 23°C. Cells were lysed by sonication in 80 mL HEGX with complete (Roche) protease inhibitors and the lysate was pelleted. Ten percent neutralized PEI (Sigma P3143) was added to the supernatant and the precipitate was removed by centrifugation. The protein was purified on a 10-mL chitin column (NEB) at 0.4 mL/min in HEGX with complete protease inhibitors. After washing, DTT was used to cleave the Tn5 transposase from the intein and eluted. The eluted protein was verified on the gel but there was an additional band. The protein was dialyzed versus 2× Tn5 dialysis buffer. After dialysis, the protein concentration was measured and used in transposome assembly with the PCR-amplified transposon from pCKD100. The transposome was transformed into electrocompetent E. tracheiphila cultures and selected on media supplemented with kanamycin and chloramphenicol together and separately. We did not obtain any colonies after repeated attempts. Modification of pCKD100 with sacB After failing to successfulyreconstitute the transposome in vitro for in vivo transposition, we tried the approach of modifying the transposon vector by cloning sacB gene into the vector outside of the transposon ends. sacBis aB. subtilisgene encoding levansucrase enzyme that converts sucrose to levans, which is harmful to the bacteriaThe gene is therefore toxic to enterobacteriaceae when grown in high concentration sucrose. To do this, the sacB gene was digested out of the plasmic, pKOBEG-sacB with the restriction enzyme NdeI. The transposon vector, pCKD100 was digested with the enzyme SphI and the linearized fragment was treated with the oligonucleotide adapter, 5' TACATG 3'. This adapter converted the SphI cohesive ends of the fragment into an NdeI ends and therefore compatible with the sacB fragment. The ligated product was transformed into R6K-proficient E. coli strain, S17-1Pir and selected on Chloramphenicol-supplemented media. Clones were grown on medium with 10% sucrose to ensure they did not grow. These clones are presently being confirmed with mutagenesis in other Enterobacteriaceae and later with sequencing.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Nazareno, E. S., Kersey, C. M. and Dumenyo, C. K. 2016. Characterization of the incompatible interaction between Erwinia tracheiphila and non-host tobacco (Nicotiana tabacum). Physiological and Molecular Plant Pathology. DOI: http://dx.doi.org/10.1016/j.pmpp.2016.10.001. URL: http://www.sciencedirect.com/science/article/pii/S0885576516301448
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Rabiul Islam, Shyretha Brown and C. Korsi Dumenyo. 2016. NAD-dependent epimerase/dehydratase affects cell surface properties, virulence and extracellular enzyme production in the soft rot phytopathogen, Pectobacterium carotovorum. Tennessee State University Research Symposium
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Eric S. Nazareno, Caleb M. Kersey, and C. Korsi Dumenyo. 2016. Characterization of the incompatible interaction between Erwinia tracheiphila and non-host tobacco (Nicotiana tabacum). Tennessee State University Research Symposium.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
U. Adhikari* and C. K. Dumenyo. 2016. rsmK mutant of the soft-rotting enterobacterial phytopathogen, Pectobacterium carotovorum, elicits hypersensitive response in tobacco in addition to overproduction of plant cell wall-degrading enzymes. Tennessee State University Research Symposium. Abstract number GR SCI 46
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2016
Citation:
Rabiul Islam, 2016. NAD-dependent epimerase/dehydratase affects cell surface properties, virulence and extracellular enzyme production in the soft rot phytopathogen, Pectobacterium carotovorum. MS Thesis. Tennessee State University.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2016
Citation:
Eric S. Nazareno. 2016. Characterization of host and non-host interactions of the bacterial wilt pathogen, Erwinia tracheiphila. MS Thesis. Tennessee State University.
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Progress 01/09/15 to 09/30/15
Outputs
Target Audience:The target audince for this project is the agricultural, research, extension and education community in the US and across the world. Changes/Problems:We encouted a problem with the generation of transposon insertion mutants using the conditional replication origin (R6K)-basedplasmid. Instead of the plasmid acting as a suicide vector, it is able to replicate in E. tracheiphila. We are considering alternate approaches for generating the mutants. What opportunities for training and professional development has the project provided?The project has provided for the training of one graduate student. He has been employed 50% as a graduate research assistant on this project while working on his MS degree in Agricultural Sciences.He has had he opportunity to present his findings in three local meeting:- University-wide research symposium, Annual meeting of Tennessee/Kentucky branch of American Society for Microbiology and the annual meeting of the Tennessee Academy of Science. Through the project, laboratory research experientialtraining was provided toa high school summer intern for five weeks. The student worked with the graduate student on pathogen tracking in infected tissues. The high school intern enrolled as an undergraduate and continued in the laboratory as an undergraduate researcher under the Dean's Scholars Program. How have the results been disseminated to communities of interest?Findings from this project have been disseminated through 1. Publicationin peer-reviewed journal 2. Presentation in University-wide research symposium 3. Annual meetingof Tennessee Academy of Sciences 4. Annuial meeting of Tennessee/Kentucky branch of American Academy of sciences 5. Additional manuscripts are in preparation for submission. What do you plan to do during the next reporting period to accomplish the goals?We are going to over express and purify transposase TnpA protein from E. coli. This purified protein along with the transposon already constructed for GFP tagging and positive antibiotic selection will be used to establish an in vivo transposon mutagenesis system patterned on EZ-TN systemfor Erwinia tracheiphila. Once established, GFP fusion mutants will be selected under various conditions including different known inducers of virulence in other pathogenic bacterial systems.
Impacts
What was accomplished under these goals?
Transposon mutagenesis of E. tracheiphila Transposon or-based strategies are useful for determining gene functions in various organisms. In bacteria, the transposon Tn5 has been a very important tool in genetic studies. For functional genomics of E. tracheiphila, we wanted to use transposons to generate mutants in genes which respond to different signals. We constructed a new transposon vector, pTnModRCm-GFP by combining features from two vectors, pTnMod-RCm and pTGN. pTnMod-RCm-GFP has a λ-Pir-dependent R6K origin of replication, a chloramphenicol (Cm) resistance gene, and the promoterless gfp-nptII cassette to be driven by the truncated promoter. Two methods were used to deliver the transposon into E. tracheiphila. The transposon was used in bi-parental mating to mutagenize E. tracheiphila strains using the tra funcations of E. coli S17-1 λ-Pir. Electroporation was also used to deliver the plasmid into E. tracheiphila with the expectation that the R6K-based plasmid will not replicate in the E. tracheiphila host. Tranconjugants were selected thatdid not express FGP and grew only on Nalidixic acid and chloramphenicol but not kanamycin which is also promoter-less like gfp gene. Further investigation reveal the plasmid was replicative in E. tracheiphila and did not act as a suicide vector. Thus, the tranconjugants carried the whole plasmid and were not transposon mutants. This vector could therefore not be used for mutagenesis of E. tracheiphila HR survey among E. tracheiphila strains or elicitation of hysensitive response. Following assembly and preliminary annotation of the genomes of three E. tracheiphila strains from melon, cucumber and squash, we determined that these organism each have a copy of hrpN, the gene encoding harpin, the elicitor of hypersensitive response. We therefore wanted to study if E. tracheiphila strains interact with tobacco the same way that other harpin producing enterobacteriacea do. Twenty-three strains of E. tracheiphila were surveyed for elicitation of HR in tobacco. We found that 21 of of the 23 strains were able to induce HR in tobacco All tested Et strains of Cucurbita and Cucumis origin elicited HR.. From the 21 HR-inducing strains, we selected three (one from each host) for further studies. Spontaneous nalidixic acid mutants of HCa1-5, UnisCu1-1, and MISpSq were labeled with a plasmid carrying the green fluorescent protein (gfp) gene. GFP detection in planta. The three selected strains were labeled with green fluorescent protein (GFP) by transformation with the plasmid that we had constructed containing pGEMT-GfpUv. Positive transformants were selected in ampicillin (100 µg/ml) and GFP was observed using a hand-held UV lamp. The labeled strains were infiltrated in tobacco leaves using the method described above. After 24 hrs, leaves were exposed under UV light to visualize fluorescent bacteria in the infiltrated region. We observed GFP fluorescence on plate of transformed colonies. We inoculated the labeled strains to tobacco leaves and detected fluorescence at the region of infiltration after 24 hrs. Harpin sequence alignment and phylogenetic tree. Harpin sequences of E. tracheiphila HCa1-5, UnisCu1-1, and MispSq were obtained from our draft sequences (unpublished data) and aligned with Erwinia tracheiphila PSU-1 harpin (Genebank accession no. EOS94912.1) using CLUSTAL Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Multiple sequence alignment of the harpin proteins of Enterobacteriaceae was conducted using COBALT (http://www.st-va.ncbi.nlm.nih.gov/tools/cobalt). The alignment was downloaded as a Newick file then uploaded to iTOL (http://itol.embl.de/) to generate the phylogenetic tree.The alignment of HrpN protein sequences of E. tracheiphila strain using CLUSTAL Omega shows that both HCa1-5 and UnisCu1-1 (Cucumis strains) HrpN have 405 aa while MISpSq hrpN has 451 aa and PSU-1 has 454 aa (Cucurbita strains) (Fig. 2). hrpN of MISpSq, a Cucubita strain has the closest identity to hrpN of PSU-1; Cucumis strains HCa1-5 and UnisCu1-1 had 100% hrpN sequence identity. The alignment also reveals that the two groups of strains are 97% identical. Phylogenetic analysis of the hrpN sequences indicated that E. tracheiphila hrpN is closer to those from the more distantly-related Pantoea agglomerans, Pantoea stewartii, and Erwinia pyrifoliae than to E. amylovora; and less related to the soft rot bacteria, Pectobacterium spp. and Dickeya spp. (Fig. 3). Our distance tree shows four distinct groups in the Erwinias HrpN.: 1) non HR-inducing Pectobacterium spp.; 2) HR-inducing Dickeya spp.; 3) HR-inducing Pantoea spp. and E. tracheiphila; and 4) HR-inducing E. amylovora. Conductivity test. . Cultures of GFP-labeled bacteria HCa1-5N/GFP, UnisCu1-1N/GFP, and MispSq-N/GFP strains were infiltrated in tobacco leaves using the method described, with Erwinia amylovora strain E9 as the positive control and E. coli DH5α as the negative control. Five leaf discs We found an increase in ion concentrations at the leaf area of bacterial infiltration after 24 hrs . Statistical analysis showed that HCa1-5N/GFP, UnisCu1-1N/GFP and MISpSq-N/GFP induced leaf conductivity levels that are not significantly different from E. amylovora E9, a classical inducer of HR that was included as the positive control. The leaf areas infiltrated with E. coli DH5α and distilled water had significantly lower conductivity. Prevention of HR. Another hallmark characteristic of hypersensitive response in plants induced by phytobacteria is the ability of lower concentrations of the inoculum to prevent HR when infiltrated into a leaf prior to a challenge by HR-inducing concentration of the HR-inducing organism. To show that the reaction elicited by E. tracheiphila strains in tobacco is classical HR, we tested the ability of lower concentration inoculum of E. trachiphila strains to prevent HR when pre-infiltrated into the challenged leaf area. We pre-infiltrated tobacco leaves with lower concentrations of inoculum then followed up with a challenge with HR-inducing concentrations at the same sites of preinfiltration after 24 hrs. The pre-infiltrated low concentration inoculum showed no HR while the HR-inducing concentrations infiltrated at the same time exhibited programmed cell death. We observed that HR was prevented in the pre-infiltrated regions when these same areas were later challenged. The same inoculum that failed to elicit HR in preinfiltrated area elicited HR in areas of the same leaf that were not preinfiltrated. This indicated E. tracheiphila elicits classical hypersensitive response. Bacterial population count. We infiltrated HR-inducing concentrations of bacteria into tobacco leaves and recovered the bacterial cells from the leaves. HCa1-5N/GFP, UnisCu1-1N/GFP, and MISpSq-N/GFP were infiltrated in tobacco leaves. After infiltration, 4 leaf discs were collected from the infiltrated area and into 500 µl water in 2ml microsentrifuge tubes. A 5 mm stainless steel bead was placed in each tube and the leaf discs were ground for 2 mins at 25 Hz using Tissue Lyser II (Qiagen, CA, USA). After grinding, the volume was raised to 1ml and serial dilution was done to 10-3. From each dilution, 100 µl was spread on NY plates with nalidixic acid (25 µg/ml). Cell counts were obtained at Day 0, 1, and 3. The experiment was done in 3-4 replicates. Colony counts showed that the bacterial populations in the infiltrated area were decreased from Day 0 to Day 3 post inoculation. The experiment was conducted twice with at least two replications. These data clearly show that E. tracheiphila strains do elicit classical HR.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Nazareno ES and Dumenyo CK. 2015. Modified inoculation and disease assessment methods reveal host specificity in Erwinia tracheiphila-Cucurbitaceae interactions. Microbial Pathogenesis 89:184-7. doi: 10.1016/j.micpath.2015.10.013. Epub 2015 Oct 30.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
U. Adhikari and C. Korsi Dumenyo. 2015. Inactivation of rsmK of the soft-rotting enterobacterial phytopathogen, Pectobacterium carotovorum, leads to overproduction of plant cell wall-degrading enzymes and hypervirulence. Presented at Annual Meeting of Tennessee Academy of Sciences. Middle Tennessee State University.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Eric Nazareno and C. Korsi Dumenyo. 2015. A multi-feature transposon for genetic studies of human- and plant-pathogenic bacteria. Presented at the Annual Meeting of TN/KY Branch of American Society for Microbiology. Middle TN State University.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
E. Nazareno and CK Dumenyo. 2015. Construction of a GFP-based and positive antibiotic selection transposon for genetic studies in phytopathogenic Enterobacteriaceae. Unviersity-Wide Research Symposium, Tennessee State University.
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