Presentation Title

Transposon Mutagenesis ni DNA-binding protein H-NS Affects EPS Expression and Motility in Paraburkholderia unamae

Faculty Mentor

Shelley Thai

Start Date

18-11-2017 9:59 AM

End Date

18-11-2017 11:00 AM

Location

BSC-Ursa Minor 64

Session

Poster 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Paraburkholderia unamae is a symbiotic, nitrogen-fixing bacteria that has been shown to promote plant growth. The expression of exopolysaccharide (EPS) and motility are thought to serve as important factors in mediating the symbiotic relationship between P. unamae and its host plant. The focus of this study was to identify genes that regulate the production of EPS and/or expression of motility. We then analyzed the genes in order to understand how they achieved regulation of these phenotypes. P. unamae underwent conjugation with E. coli BW20767 on tryptone yeast agar (TY) medium to facilitate the transfer of the Tn5-RL27 suicide vector containing a kanamycin (kan) resistance gene. The P. unamae mutants containing the Tn5-RL27 transposable element were selected on M9 media containing rifampicin and kan. 1200 of the resulting colonies where screened for abnormalities in EPS production and motility expression. DNA from selected mutants was isolated and cut using restriction enzymes to retrieve the transposable element along with the DNA region into which it inserted. The disrupted gene was sequenced, then identified and analyzed using BLAST on the NCBI and IMG/MER databases. Six mutants were found to have the transposable element within or just upstream the hns gene. All six mutants exhibited an overproduction of EPS. Five of the mutants also showed highly reduced motility when grown in soft agar. The hns gene codes for the histone-like nucleoid-structuring (H-NS) protein. The H-NS protein is known to act as a pleiotropic, global regulator in Echerichia coli. H-NS positively regulates the biogenesis of the flagella in E. coli, and has been shown to negatively regulate EPS production in Vibrio cholerae. Although hns has not previously been studied in P. unamae, it seems to serve an equally important role in this organism’s motility and EPS production and perhaps other cellular processes.

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Nov 18th, 9:59 AM Nov 18th, 11:00 AM

Transposon Mutagenesis ni DNA-binding protein H-NS Affects EPS Expression and Motility in Paraburkholderia unamae

BSC-Ursa Minor 64

Paraburkholderia unamae is a symbiotic, nitrogen-fixing bacteria that has been shown to promote plant growth. The expression of exopolysaccharide (EPS) and motility are thought to serve as important factors in mediating the symbiotic relationship between P. unamae and its host plant. The focus of this study was to identify genes that regulate the production of EPS and/or expression of motility. We then analyzed the genes in order to understand how they achieved regulation of these phenotypes. P. unamae underwent conjugation with E. coli BW20767 on tryptone yeast agar (TY) medium to facilitate the transfer of the Tn5-RL27 suicide vector containing a kanamycin (kan) resistance gene. The P. unamae mutants containing the Tn5-RL27 transposable element were selected on M9 media containing rifampicin and kan. 1200 of the resulting colonies where screened for abnormalities in EPS production and motility expression. DNA from selected mutants was isolated and cut using restriction enzymes to retrieve the transposable element along with the DNA region into which it inserted. The disrupted gene was sequenced, then identified and analyzed using BLAST on the NCBI and IMG/MER databases. Six mutants were found to have the transposable element within or just upstream the hns gene. All six mutants exhibited an overproduction of EPS. Five of the mutants also showed highly reduced motility when grown in soft agar. The hns gene codes for the histone-like nucleoid-structuring (H-NS) protein. The H-NS protein is known to act as a pleiotropic, global regulator in Echerichia coli. H-NS positively regulates the biogenesis of the flagella in E. coli, and has been shown to negatively regulate EPS production in Vibrio cholerae. Although hns has not previously been studied in P. unamae, it seems to serve an equally important role in this organism’s motility and EPS production and perhaps other cellular processes.