An MTA phosphorylase gene discovered in the metagenomic library derived from Antarctic top soil during screening for lipolytic active clones confers strong pink fluorescence in the presence of rhodamine B.
In this work, we present the construction of a metagenomic library in Escherichia coli using the pUC19 vector and environmental DNA directly isolated from Antarctic topsoil and screened for lipolytic enzymes. Unexpectedly, the screening on agar supplemented with olive oil and rhodamine B revealed one unusual pink fluorescent clone (PINKuv) out of 85 000 clones. This clone harbored a plasmid, pPINKuv, which has an insert of 8317 bp that has been completely sequenced. Further analysis of the insert showed eight ORFs. Three ORFs among these exhibited similarities to Psychrobacter arcticus genes. A nucleotide sequence designated as ORF4 encoded a protein with 93% identity to the methylthioadenosine phosphorylase of P. arcticus. This protein was responsible for the observed pink fluorescence of the PINKuv clone in the presence of rhodamine B. We found that colonies of recombinant E. coli TOP10F'/pUC19-ORF4 strain showed pink fluorescence under UV illumination on the Luria-Bertani agar supplemented with rhodamine B after culturing at 25, 30 and 37 degrees C. The same effect was achieved using other E. coli strains such as DH5alpha, LMG194, JM101 and BL21(DE3) pLysS. The results presented here will provide the basis for further studies on the use of the discovered gene as a new reporter gene for molecular biology applications.
Cieśliński
Hubert
H
Department of Microbiology, Chemical Faculty, Gdańsk University of Technology, Narutowicza 11/12, Gdańsk, Poland. hcieslin@pg.gda.pl
Długołecka
Anna
A
Kur
Józef
J
Turkiewicz
Marianna
M
D000864:Antarctic Regions; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D008969:Molecular Sequence Data; D000069463:Olive Oil; D016366:Open Reading Frames; D010860:Pigments, Biological / Q000378:metabolism*; D010938:Plant Oils / Q000378:metabolism; D044096:Psychrobacter / Q000235:genetics; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D012988:Soil Microbiology
D000069463:Olive Oil; D010860:Pigments, Biological; D010938:Plant Oils; D012235:Rhodamines; D004247:DNA; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase; C029773:rhodamine B