Presentation Title

Paraburkholderia unamae Mutants Generated By Transposon Mutagenesis Show Irregular EPS Expression and Motility

Faculty Mentor

Dr. Shelley Thai

Start Date

18-11-2017 2:15 PM

End Date

18-11-2017 3:15 PM

Location

BSC-Ursa Minor 65

Session

Poster 3

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

The goal of this study was to map out genes of the diazotrophic bacterium, Paraburkholderia unamae, and further access the function of those genes involved in exopolysaccharide (EPS) production and motility, the two phenotypes associated with the bacterium’s method of symbiosis. Paraburkholderia unamae forms commensalistic relationships with coffee, maize, and sugarcane; the bacteria’s ability to fix nitrogen for these crops show promise for possible enhanced fertilizers once P. unamae is better accessed. To map out which genes are involved in EPS and motility P. unamae was randomly mutated in this study using transposon mutagenesis. The plasmid that we used, pRL27, contains the Tn5 transposable element, which was used to mutate P. unamae. 400 colonies that contain the transposon were screened for EPS and motility mutants. The DNA of two promising mutants (AAD21 and TR414) containing mutations affecting EPS Production and motility were then isolated and sequenced. Mutations were found interfering in flagellar rotary genes. These findings suggest that there may be complex systems in play that compensate for the lack of motility through EPS production. To better understand polar effects that these mutations may have, RT-PCR could be used to identify which genes are affected, thus allowing us to focus on these particular genes for further studies.

Summary of research results to be presented

Both mutants in this study showed an overexpression of EPS and were non-motile compared to the wildtype. The mutants’ sequences were analyzed on BLAST and the transposons were found in neighborhoods containing genes involved in flagellar biosynthesis. The AAD21 mutation was inserted directly into the fliG gene, part of the fliHGF that codes for the base rotor-mounted switch complex (C ring) of the basal body. This complex interacts with chemotaxis proteins which determine the direction of flagellar rotation. The TR414 mutation was found in the intergenic region surrounded by the genes that code for the L and P ring proteins, which are involved in providing smooth rotations for the flagella. Additionally, TR-414 contains a hypothetical protein downstream of from the mutation whose function is unknown.

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Nov 18th, 2:15 PM Nov 18th, 3:15 PM

Paraburkholderia unamae Mutants Generated By Transposon Mutagenesis Show Irregular EPS Expression and Motility

BSC-Ursa Minor 65

The goal of this study was to map out genes of the diazotrophic bacterium, Paraburkholderia unamae, and further access the function of those genes involved in exopolysaccharide (EPS) production and motility, the two phenotypes associated with the bacterium’s method of symbiosis. Paraburkholderia unamae forms commensalistic relationships with coffee, maize, and sugarcane; the bacteria’s ability to fix nitrogen for these crops show promise for possible enhanced fertilizers once P. unamae is better accessed. To map out which genes are involved in EPS and motility P. unamae was randomly mutated in this study using transposon mutagenesis. The plasmid that we used, pRL27, contains the Tn5 transposable element, which was used to mutate P. unamae. 400 colonies that contain the transposon were screened for EPS and motility mutants. The DNA of two promising mutants (AAD21 and TR414) containing mutations affecting EPS Production and motility were then isolated and sequenced. Mutations were found interfering in flagellar rotary genes. These findings suggest that there may be complex systems in play that compensate for the lack of motility through EPS production. To better understand polar effects that these mutations may have, RT-PCR could be used to identify which genes are affected, thus allowing us to focus on these particular genes for further studies.