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Complete genome sequencing of Pandoraea pnomenusa RB38 and Molecular Characterization of Its N-acyl homoserine lactone synthase gene ppnI.

Lim YL, Ee R, How KY, Lee SK, Yong D, Tee KK, Yin WF, Chan KG - PeerJ (2015)

Bottom Line: A pair of cognate luxI/R homologs was identified where the luxI homolog, ppnI, was found adjacent to a luxR homolog, ppnR1.Burkholderia spp.To our knowledge, this is the first documentation of the luxI/R homologs of the genus Pandoraea.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Genetics and Molecular Biology, Faculty of Science, Institute of Biological Sciences, University of Malaya , Kuala Lumpur , Malaysia.

ABSTRACT
In this study, we sequenced the genome of Pandoraea pnomenusa RB38 using Pacific Biosciences RSII (PacBio) Single Molecule Real Time (SMRT) sequencing technology. A pair of cognate luxI/R homologs was identified where the luxI homolog, ppnI, was found adjacent to a luxR homolog, ppnR1. An additional orphan luxR homolog, ppnR2, was also discovered. Multiple sequence alignment and phylogenetic analysis revealed that ppnI is an N-acyl homoserine lactone (AHL) synthase gene that is distinct from those of the nearest phylogenetic neighbor viz. Burkholderia spp. High resolution tandem mass spectrometry (LC-MS/MS) analysis showed that Escherichia coli BL21 harboring ppnI produced a similar AHL profile (N-octanoylhomoserine lactone, C8-HSL) as P. pnomenusa RB38, the wild-type donor strain, confirming that PpnI directed the synthesis of AHL in P. pnomenusa RB38. To our knowledge, this is the first documentation of the luxI/R homologs of the genus Pandoraea.

No MeSH data available.


Related in: MedlinePlus

TLC bioassay overlaid with CV026 biosensor.Lane A: AHL extract of E. coli BL21(DE3)pLysS::ppnI; Lane B: Synthetic C8-HSL.
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fig-5: TLC bioassay overlaid with CV026 biosensor.Lane A: AHL extract of E. coli BL21(DE3)pLysS::ppnI; Lane B: Synthetic C8-HSL.

Mentions: For functional studies, we cloned the putative ppnI into a pGS-21a expression vector and subsequently transformed the pGS-21a::ppnI plasmid into competent E. coli BL21(DE3)pLysS. AHL screening were performed using C. violaceum CV026 biosensor with E. coli BL21(DE3)pLysS::ppnI. The result of the cross-streak bioassay demonstrated activation of purple violacein secretion of C. violaceum CV026 (Fig. 4A) as well as bioluminescence activity of E. coli [pSB401] indicating the production of short chain AHLs by the ppnI gene (Fig. 4B). Besides that, formation of a sole purple violacein spot on CV026 lawn which corresponds to the same retention time of the synthetic C8-HSL suggested that the ppnI is responsible for the production of C8-HSL in P. pnomenusa RB38 (Fig. 5). The AHL profile of ppnI was further verified using LC-MS/MS mass spectrometry system and only C8-HSL was detected in the supernatant of recombinant E. coli BL21 suggesting that ppnI is indeed the functional LuxI synthase of P. pnomenusa RB38 (Fig. 6).


Complete genome sequencing of Pandoraea pnomenusa RB38 and Molecular Characterization of Its N-acyl homoserine lactone synthase gene ppnI.

Lim YL, Ee R, How KY, Lee SK, Yong D, Tee KK, Yin WF, Chan KG - PeerJ (2015)

TLC bioassay overlaid with CV026 biosensor.Lane A: AHL extract of E. coli BL21(DE3)pLysS::ppnI; Lane B: Synthetic C8-HSL.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556143&req=5

fig-5: TLC bioassay overlaid with CV026 biosensor.Lane A: AHL extract of E. coli BL21(DE3)pLysS::ppnI; Lane B: Synthetic C8-HSL.
Mentions: For functional studies, we cloned the putative ppnI into a pGS-21a expression vector and subsequently transformed the pGS-21a::ppnI plasmid into competent E. coli BL21(DE3)pLysS. AHL screening were performed using C. violaceum CV026 biosensor with E. coli BL21(DE3)pLysS::ppnI. The result of the cross-streak bioassay demonstrated activation of purple violacein secretion of C. violaceum CV026 (Fig. 4A) as well as bioluminescence activity of E. coli [pSB401] indicating the production of short chain AHLs by the ppnI gene (Fig. 4B). Besides that, formation of a sole purple violacein spot on CV026 lawn which corresponds to the same retention time of the synthetic C8-HSL suggested that the ppnI is responsible for the production of C8-HSL in P. pnomenusa RB38 (Fig. 5). The AHL profile of ppnI was further verified using LC-MS/MS mass spectrometry system and only C8-HSL was detected in the supernatant of recombinant E. coli BL21 suggesting that ppnI is indeed the functional LuxI synthase of P. pnomenusa RB38 (Fig. 6).

Bottom Line: A pair of cognate luxI/R homologs was identified where the luxI homolog, ppnI, was found adjacent to a luxR homolog, ppnR1.Burkholderia spp.To our knowledge, this is the first documentation of the luxI/R homologs of the genus Pandoraea.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Genetics and Molecular Biology, Faculty of Science, Institute of Biological Sciences, University of Malaya , Kuala Lumpur , Malaysia.

ABSTRACT
In this study, we sequenced the genome of Pandoraea pnomenusa RB38 using Pacific Biosciences RSII (PacBio) Single Molecule Real Time (SMRT) sequencing technology. A pair of cognate luxI/R homologs was identified where the luxI homolog, ppnI, was found adjacent to a luxR homolog, ppnR1. An additional orphan luxR homolog, ppnR2, was also discovered. Multiple sequence alignment and phylogenetic analysis revealed that ppnI is an N-acyl homoserine lactone (AHL) synthase gene that is distinct from those of the nearest phylogenetic neighbor viz. Burkholderia spp. High resolution tandem mass spectrometry (LC-MS/MS) analysis showed that Escherichia coli BL21 harboring ppnI produced a similar AHL profile (N-octanoylhomoserine lactone, C8-HSL) as P. pnomenusa RB38, the wild-type donor strain, confirming that PpnI directed the synthesis of AHL in P. pnomenusa RB38. To our knowledge, this is the first documentation of the luxI/R homologs of the genus Pandoraea.

No MeSH data available.


Related in: MedlinePlus