Presentation Title

Over EPS Production and Decreased Motility as a Result of Transposon Mutagenesis in Paraburkholderia unamae

Faculty Mentor

Shelley Thai

Start Date

17-11-2018 8:30 AM

End Date

17-11-2018 10:30 AM

Location

CREVELING 30

Session

POSTER 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Over EPS Production and Decreased Motility as a Result of Transposon Mutagenesis in Paraburkholderia unamae

Nickolas Yedgarian1, Ania Shirvanian1, Dr. Michelle R. Lum*, and Dr. Shelley Thai1

1Glendale Community College, Biology Division, 1500 N. Verdugo Rd., Glendale, CA 91208 *Loyola Marymount University, Department of Biology, 1 LMU Dr. Los Angeles, CA 90045

Intro: The basis for this study is on the coccobacillus, gram-negative bacterium Paraburkholderia unamae. This species of bacteria is within the monophyletic group Paraburkholderia, signifying its characteristic of being nonpathogenic. P. unamae is known for its ability to fix atmospheric nitrogen gas in a symbiotic relationship with plants, and can be found in sugarcane, coffee, and maize plants (Caballero-Mellado et al., 2004). Objective: The goal of this project was to mutate P. unamae by disrupting genes that regulate EPS (exopolysaccharide) production and motility. This was done through the transposable element Tn5-RL27. Methods: Colonies of Escherichia coli carrying Tn5-RL27 were conjugated with P. unamae. Selection and screening took place to narrow down mutants of interest. Genomic DNA of P. unamae carrying the Tn5 insertion was isolated and digested. Ligation product of digested samples were then transformed into competent E. coli. The plasmids were then isolated and sequenced to determine the location of Tn5 insertion. Results: The mutants produced were AS A-12 and NY 1.36: both demonstrated overexpression of EPS and a reduction in motility. The reduction in motility was a 70% and 60.8% decrease for AS A-12 and NY 1.36, respectively. The AS A-12 mutant had a disruption in the gene ATP-dependent proteinase, coding for intracellular proteolysis while NY 1.36 mutant had a disruption in a gene known as S-formylglutathione hydrolase FrmB whose gene product is involved in formaldehyde detoxification. Conclusion: Both genes affected are involved in EPS production and motility for P. unamae.

Summary of research results to be presented

  • Mutants AS A-12 and NY 1.36 show an increase in EPS production.

  • Remarkable decrease in motility in both mutants.

  • Transposons interrupted/affected genes that encode for enzymes in both mutants.

  • AS A-12 had a diameter of 3.0mm, and NY 1.36 had a diameter of 4.0mm. Compared to wild-types 1 and 2 (10.0mm and 10.2mm, respectively), there is a clear difference in motility levels in the mutants (two wild-types present due to data being collected from different experiments). A 70% decrease for AS A-12 and a 60.8% decrease for NY 1.36.

  • AS A-12 showcased a disruption within the gene ATP-dependent proteinase, responsible for intracellular proteolysis.

  • NY 1.36 had the transposon inserted into the intergenic region in the reverse direction, possibly affecting the gene S-formylglutathione hydrolase FrmB which is responsible for formaldehyde detoxification.

  • The AS A-12 mutant had a disruption in the gene ATP-dependent proteinase, coding for intracellular proteolysis while NY 1.36 mutant had a disruption in a gene known as S-formylglutathione hydrolase FrmB whose gene product is involved in formaldehyde detoxification.

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Nov 17th, 8:30 AM Nov 17th, 10:30 AM

Over EPS Production and Decreased Motility as a Result of Transposon Mutagenesis in Paraburkholderia unamae

CREVELING 30

Over EPS Production and Decreased Motility as a Result of Transposon Mutagenesis in Paraburkholderia unamae

Nickolas Yedgarian1, Ania Shirvanian1, Dr. Michelle R. Lum*, and Dr. Shelley Thai1

1Glendale Community College, Biology Division, 1500 N. Verdugo Rd., Glendale, CA 91208 *Loyola Marymount University, Department of Biology, 1 LMU Dr. Los Angeles, CA 90045

Intro: The basis for this study is on the coccobacillus, gram-negative bacterium Paraburkholderia unamae. This species of bacteria is within the monophyletic group Paraburkholderia, signifying its characteristic of being nonpathogenic. P. unamae is known for its ability to fix atmospheric nitrogen gas in a symbiotic relationship with plants, and can be found in sugarcane, coffee, and maize plants (Caballero-Mellado et al., 2004). Objective: The goal of this project was to mutate P. unamae by disrupting genes that regulate EPS (exopolysaccharide) production and motility. This was done through the transposable element Tn5-RL27. Methods: Colonies of Escherichia coli carrying Tn5-RL27 were conjugated with P. unamae. Selection and screening took place to narrow down mutants of interest. Genomic DNA of P. unamae carrying the Tn5 insertion was isolated and digested. Ligation product of digested samples were then transformed into competent E. coli. The plasmids were then isolated and sequenced to determine the location of Tn5 insertion. Results: The mutants produced were AS A-12 and NY 1.36: both demonstrated overexpression of EPS and a reduction in motility. The reduction in motility was a 70% and 60.8% decrease for AS A-12 and NY 1.36, respectively. The AS A-12 mutant had a disruption in the gene ATP-dependent proteinase, coding for intracellular proteolysis while NY 1.36 mutant had a disruption in a gene known as S-formylglutathione hydrolase FrmB whose gene product is involved in formaldehyde detoxification. Conclusion: Both genes affected are involved in EPS production and motility for P. unamae.