Progress 02/02/18 to 09/30/20
Outputs
Target Audience:The target audience of this project is the agricultural research and education community including, researchers, professors, teachers, high school, undergraduate and graduate students, agricultural extension, and agricultural policy making community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate and two undergraduate students were trained under this project. One graduate student finished his project and earned his MS degree in summer 2019 and is employed in the Biotech industry. The second graduate student will complete a semester after the end of this project and move on to a doctoral degree program. Both undergraduates have graduated; one is now in graduate school and the other is employed. How have the results been disseminated to communities of interest?1.The results are beingincorporated into the classes that the project director teaches. 2. The results have been presented in conferences including Tennessee State University Research Symposium, Branch meeting of Kentucky/Tennessee branchof American Society for Microbiology, Southern Division Meeting of American Phytopathological Society and Biennial International Scientific Conference of Association of Nepalese AgriculturalProfessionals of America. 3. The results have been publishead in peer reviewed journals including Microorganisms, Horticulturae, Letters in Applied Micriobiology, and Jornal of Phytopathology. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Most of the effort in final year of the projectwas directed at the accomplishment of objective 4 namely, identification of virulence associated genes affected in the mutants, and therole of these genesin pathogenesis ofPectobacterium carotovorawhich has been renames P. versatile. During the previous reporting periods, we isolated in 19 mutants of Pectobacterium versatile using strain KD100 from the mutant libraty generated in the previous periods. These mutants were altered in the level of extracellular enzyme production. Compared to the parental strain KD100, three of the mutants produced less enzymes while the remaining 16 produced more enzymes than the parent. The enzymes measured include pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The level of the enzymes were measured qualitatively, semi-quantitatively and quantitatively. Qualitatively, the activity was detected incultures growing in substrate-supplemented agar media. Semi-quantitatively, enzyme activity was measured with culture supernatants on substrate-supplemented agar-test plates. Pectate lyase and protease activities werequantitatively detected spectrophotometrically. Since the transposon vector also contain a promoter-less GFP reporter gene, the mutants we also tested for the expression of GFP reporter using fluorescence spectroscopy. All the mutants produced varying levels of GFP indicating thatthe insertion of the transposon was downstream of the promoter of the affected gene and in the same orientation. The level of fluorescence ranged from about twice abovethe parental(background) levels to several thousands. In addition to differences in the insertion sites, the variations could indicatedifferent levels of expression of the affected genes. We wanted to identify the genes whose mutations resulted in these altered phenotypes. Havingfailed using theapproaches of plasposon cloning and arbitrary primed PCR, we decided to use direct Sanger sequencing across the transposon junctions using the mutant genomic DNA. The mutant genomic DNA was purified and partially digested with the restriction enzyme Sau3A. The digests yielded high molecular weight DNA that was purified fromagarosegel. The purified partially digested DNA was used as template insequencing reaction using primers designed for the transposon sequence that could sequence across the junction into flanking genomic DNA. The sequencing reactions werepurified and analyzed using capillary sequencers. Sequences obtained were analyzed using database search against the genomic sequence of Ecc71 obtained earlier in the project. Themutant genewas identified in each case. Some of the mutants we affected in the same gene including the well characterized global regulators of virulence, rsmA and rsmC. Other mutants were affected in blaOXA2, traK, htrA and degQ genes. Analyses are ongoing on the role of the last two genes, htrA and degQ on extracellular enzyme production and virulence of the soft-rotting pathogen, Pectobacterium versatile.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Nazareno, E.S.; Acharya, B.; Dumenyo, C.K. A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria. Letters in Applied Microbiology 2020, Published online November 2020, doi:https://doi.org/10.1111/lam.13423.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Kabir, M.N.; Taheri, A.; Dumenyo, C.K. Development of PCR-Based Detection System for Soft Rot Pectobacteriaceae Pathogens Using Molecular Signatures. Microorganisms 2020, 8, doi:10.3390/microorganisms8030358.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Agyemang, P.A.; Kabir, M.N.; Kersey, C.M.; Dumenyo, C.K. The Bacterial Soft Rot Pathogens, Pectobacterium carotovorum and P. atrosepticum, Respond to Different Classes of Virulence-Inducing Host Chemical Signals. Horticulturae 2020, 6, 13
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Acharya, B.; Dumenyo, C.K. Characterization of hyper-virulent mutants of soft rot pathogen, Pectobacterium carotovorum In Proceedings of 2nd NAPA Biennial International Scientific Conference 2020, Virtual, Sept 25-28, 2020.
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The target audience of this project is the agricultural research and education community including, researchers, professors, teachers, high school, undergraduate and graduate students, agricultural extension, and agricultural policy making community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate and threeundergraduate students were trained under this project. One graduate student finished his project and earned his MS degree in summer 2019. The second graduate student is also working towards her MS degree. The undergraduates werestudent researchers. How have the results been disseminated to communities of interest?1.The results have been incorporated into the classes that the project director teaches. 2. The results have been presented in one conferences, the 2019 annual meeting of Kentucky-Tennessee branch of American Society for Microbiology. 3. The results have been published in peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? We will continue with the identification of the genetic location of the transposon in the mutants. That will tell us the genes whose interruption is leading to the increased virulence. Once identified, we will systematically analyze the role of those genes in the regulation of virulence in the soft rot bacteria. We will clone the wild type of the mutant genes and characterize the gene products. If needed, we will use the plasposons to transfer the mutations into other genetic backgrounds to generate double mutants to study how these newly identified genes interact with previously know virulence regulators in Pectobacterium.
Impacts
What was accomplished under these goals?
Supplementation of the transposon mutant library. We had earlier constructed a mutant library that contained approximately 10000 independent mutants. With an estimated 4300 genes in the genome, the library represented barely two insertions per gene. We therefore decided to construct a denser libary. Weconstructed a saturation library to cover at least five insertions per gene. Using the same promoter-probe gfp transposon vector, pCKD100 and E. coli S17-1 as the donor strain, and the nalidixic acid resistant KD100 as recipient, mutagenesis was performed and mutants, selectedon chloramphenicol and kanamycin medium were pooled together in a library of over 36000 independent mutants. This library will supplement the previous one and ensure at least five separate insertion in each non-essential gene. Rescreening of the library for hypervirulent mutants We plated the library on Nutrient-Gelatin agar medium. On this medium, extracellular protease activity can be detected non-destructively with a halo around the colony. The size of the halo indicates the level of protease activity. We isolated mutants that produced higher protease activity than the surrounding background levels. These mutants were characterized for the production all the plant cell wall-degrading enzymes, pectate lyase, polygalacturonase, protease and cellulase. The mutants were also characterized for their level of plant tissue maceration using celery petioles and potato tubers. All the mutants over produced PCWDE and were more virulent that their parent. The level of expression of GFP, which is an indication of the promoter activity of the truncated gene were also measured. Analysis of the mutants In order to determine the genetic location of the transposon in the mutants, we isolated the genomic DNA of the mutants and digested with the restriction enzyme ApaLI, which does not have a recognition site within the transposon in pCKD100. The digested DNA was self-ligated and electroporated into E. coli OneShot Pir1. We have isolated plasposons from the mutants and are being analyzed.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2019
Citation:
Hasan, Mohammed Raqibul, 2019.Isolation and Characterization of GFP-Tagged Transposon Mutants of Pectobacterium carotovorum subsp. carotovorum KD100, MS Thesis. Tennessee State University
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Kersey CM, Heintzman D, Dumenyo CK. Transposon insertion upstream of a putative sodium/sulphate symporter is associated with hypervirulence in the soft rot bacterium, Pectobacterium carotovorum. J Phytopathol. 2018.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
Acharya B, Hasan MR, Dumenyo CK. Construction of saturated GFP-tagged transposon Tn5 insertion mutant library of the soft rot pathogen, Pectobacterium carotovorum. 2019 Annual Meeting of Tennessee Academy of Science; 22-Nov-2019, 2019; Columbia, TN, USA.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Islam R, Brown S, Taheri A, Dumenyo CK. The Gene Encoding NAD-Dependent Epimerase/Dehydratase, wcaG, Affects Cell Surface Properties, Virulence, and Extracellular Enzyme Production in the Soft Rot Phytopathogen, Pectobacterium carotovorum. Microorganisms. 2019;7(6).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
Kersey CM, Dumenyo CK. Exploration of an unknown genes role in a hypervirulent mutant of the soft rot bacterium Pectobacterium carotovorum. Kentucky-Tennessee Branch of American Society for Microbiology - Fall Meeting - 2019; 8-9 November 2019, 2019; Nashville, TN, USA.
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Progress 02/02/18 to 09/30/18
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:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student, four undergraduates and a high school summer intern were trained under this project. The graduate student is working on this project towards his Master of Science degree in Agricultural Science. The four undergraduates are working as undergraduate student researchers where they are mentored on research practices. The high school student was a summer intern during 2018 summer. She worked in the lab getting experience on experimental research. How have the results been disseminated to communities of interest?Part of the results have been presented during the university-wide research symposium. More presentations are planned for 2019 professional meetings. What do you plan to do during the next reporting period to accomplish the goals?1. Analysis of the genomic sequence. The genomic sequence of Ecc71 will be further analyzed. The analysis will be done again using Galaxy platform hosted by PSC and other analytical tools. The number of coding sequences and their putative functions, sequence identity with each other and with genomes of other SRE strains, and the approximate number of genes that are absent or highly divergent among the SRE genomes. The genome sequence data will also be analyzed to determine the core and pan genomes of SREs. 2. Rescreen of the mutant library. We will rescreen the mutant library using flow cytometry. Mutants highly expressing the gfp gene will be sorted under each condition; minimal medium, celery extract supplemented medium and potato extract-supplemented medium. Each of these mutant pools will be grown under the other condition and plated on the opposite medium. Colonies that have lost the high level of gfp expression will be selected as differentially expressing gfp and analyzed like those that were selected in the previous screen using the UV light box. 3. Analysis of the mutants. The mutants will be classified into four categories: 1. mutants in potato extract inducible genes; 2, mutants in potato extract repressible genes; 3, mutants in celery extract inducible genes and 4, mutants in celery extract repressible genes. Each of these mutants will be characterized for their growth characteristics, exoenzyme production and pathogenesis. The mutants will be ranked and the high-ranking ones in each category will be selected for further analysis. The first step in the genetic analysis of each mutant is the identification of the mutant gene. This will be done by cloning the truncated gene on the plasposon and sequencing. Analysis of the sequence will reveal the mutant gene and whether it has previously been implicated in host interaction or pathogenesis.
Impacts
What was accomplished under these goals?
Sequencing and assembly of soft rot bacterial genome. In order to analyze the pathogenesis of the soft rot pathogen at the genome scale, we have sequenced and assembled the draft genomic sequence of the parental strain to many of the mutants and constructs in our laboratory, Ecc71. We used comparative genomic analysis of NGS-generated sequence data because it confers key advantages. Next generation sequencing allowed collection of extremely large amounts of nucleic acid sequence data of the bacteria through generation of numerous overlapping short sequence reads. These short reads can then be computer-assembled into contigs of sequence. Our lab, along with our collaborators at Pittsburgh Supercomputing Center have used the Illumina instruments platform of MiSeq to generate up to 500 million sequence reads per run of the genomes of Ecc71. Using the tools of bioinformatics available at our collaborator institution at Pittsburgh Supercomputing Center, we have assembled the genome of the Ecc71 strain. The next-generation sequencing allowed for true genome-wide analysis at the nucleotide level and also makes available the draft sequence for future studies. This is important for an organism for which there are no genome sequences. Using the assembly program Spades, the reads were assembled into a draft genome with the following features. The assembled sequence has a total length of 4,925,135 nucleotides with assembly gap length of 1296 and no gaps between scaffolds. The number of scaffolds were 24 with scaffold N50 of 856905 and L50 of 3. There were 26 contigs with Contig N50 and L50 as those of scaffold. Preliminary analysis of the draft genomes has revealed the presence of potential pathogenicity factors including host-specificity genes. Construction of saturated mutant library. Using the promoter-probe gfp transposon vector, pCKD100 and E. coli S17-1 as the donor strain, the Nalidixic acid resistant strain, KD100 was mutagenized to obtain random transposon insertion mutant library. Strain KD100 is a Lac- and NalR derivative of the sequenced strain, Ecc71. Mutants were selected on chloramphenicol medium to allow for the selection of inserts in both the coding and non-coding strands of the truncated gene. The mutants from many selection plates were pooled together to generate a mutant pool of over 10,000. In the library pool, mutants carrying the gfp gene fusion downstream of an active promoter will express the gfp-nptII gene cassette according to the strength of the promoter. After collection, the mutant library was screened for the expression of the truncated genes through the expression of gfp reporter gene. First, about 5000 mutants were arrayed in 48 grid agar plates. The cultures were replica-plated in minimal medium with and without host (potato or celery) extracts. Mutants were screened under UV light box for the level of fluorescence. Mutants that differentially express gfp between host-extract supplemented or un-supplemented medium were selected for further analysis.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Dumenyo, CK & Hasan, MR. 2018. Isolation of transposon mutant of Pectobacterium carotovorum in host signal regulated genes. Oral Presentation at University-wide Research Sumposium, Tennessee State University, Nashville, TN
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