Presentation Title

Disruption of LuxR Promoter Induce Quorum Sensing Response in P. unamae

Faculty Mentor

Dr. Shelley Thai

Start Date

17-11-2018 8:30 AM

End Date

17-11-2018 10:30 AM

Location

CREVELING 33

Session

POSTER 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Paraburkholderia unamae is an endophytic, Gram negative bacterium capable to facilitate plant growth by means of nitrogen fixation and lowering ethylene levels in plants. Further, P. unamae has been indicated to encode cell density-dependent regulatory genes, known as quorum sensing system, as well as virulence control, and phenol and benzene degradation genes. If transposon mutagenesis is performed in P. unamae, the genes at the site of insertion of the transposon result in mutations that are evidenced by the alteration of phenotypes like exopolysaccharide (EPS) production and motility. We tested this hypothesis by performing transposon mutagenesis using the plasmid pRL27 which carries the transposable element Tn5pRL27, and further characterizing the gene expression of P. unamae. pRL27 was transferred from E. coli to P. unamae by conjugation. Conjugants were then screened for alteration in EPS expression and motility by selecting 450 colonies to streak and poke in suitable media. Restriction digest of isolated genomic DNA from selected P. unamae mutants using Sac II enzyme was performed. Ligation of DNA fragments was made and then transformed into E. coli. Plasmids containing the transposon were isolated and sequenced to determine the genomic region of Tn5 disruption. After sequencing, a gene map of the site of insertion of the transposon in the genome of the selected mutant AM4.186 was proposed, enabling to determine its affected genes and respective functions. Further, mutant AM4.186 showed significant reduced motility when compared to the wild type organism. The site of insertion of the transposable element in mutant AM4.186 indicated the disruption of the promoter region of two overlapping quorum sensing transcriptional regulator genes (histKA-luxR) inducing EPS overproduction. Reduced motility and over EPS production in mutant AM4.186 which favor the formation of biofilms, agree with the quorum sensing response coded in the luxR-like gene of P. unamae.

Summary of research results to be presented

Mutant AM4.186 showed significant reduced motility when compared to the wild type organism, with 51.7% reduction. Also, mutant AM 4.186 was observed to be an over EPS producer when compared to the wild type organism. After obtaining DNA sequences, BLAST analysis indicated that for mutant AM 4.186 the transposon inserted in the promoter region of the quorum sensing transcriptional regulator genes histKA and luxR. To complement the performed qualitative analysis of over EPS production, quantitative RT-PCR can be used to measure the transcription level of histKA-luxR genes. The protein levels expressed by histKA-luxR genes can be determined with western blot.

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

Disruption of LuxR Promoter Induce Quorum Sensing Response in P. unamae

CREVELING 33

Paraburkholderia unamae is an endophytic, Gram negative bacterium capable to facilitate plant growth by means of nitrogen fixation and lowering ethylene levels in plants. Further, P. unamae has been indicated to encode cell density-dependent regulatory genes, known as quorum sensing system, as well as virulence control, and phenol and benzene degradation genes. If transposon mutagenesis is performed in P. unamae, the genes at the site of insertion of the transposon result in mutations that are evidenced by the alteration of phenotypes like exopolysaccharide (EPS) production and motility. We tested this hypothesis by performing transposon mutagenesis using the plasmid pRL27 which carries the transposable element Tn5pRL27, and further characterizing the gene expression of P. unamae. pRL27 was transferred from E. coli to P. unamae by conjugation. Conjugants were then screened for alteration in EPS expression and motility by selecting 450 colonies to streak and poke in suitable media. Restriction digest of isolated genomic DNA from selected P. unamae mutants using Sac II enzyme was performed. Ligation of DNA fragments was made and then transformed into E. coli. Plasmids containing the transposon were isolated and sequenced to determine the genomic region of Tn5 disruption. After sequencing, a gene map of the site of insertion of the transposon in the genome of the selected mutant AM4.186 was proposed, enabling to determine its affected genes and respective functions. Further, mutant AM4.186 showed significant reduced motility when compared to the wild type organism. The site of insertion of the transposable element in mutant AM4.186 indicated the disruption of the promoter region of two overlapping quorum sensing transcriptional regulator genes (histKA-luxR) inducing EPS overproduction. Reduced motility and over EPS production in mutant AM4.186 which favor the formation of biofilms, agree with the quorum sensing response coded in the luxR-like gene of P. unamae.