Journal: Nature microbiology
Article Title: A metabolic pathway for catabolizing levulinic acid in bacteria
doi: 10.1038/s41564-017-0028-z
Figure Lengend Snippet: a , Organization of the lvaRABCDEFG (9,323 bp) operon. b , Reverse Transcriptase (RT) PCR demonstrates that each gene is expressed in cells grown on LA. Samples were compared with the negative control (-RT) where reverse transcriptase was omitted from the reaction ( n =1). c , RT-PCR of cDNA created with primer JMR237 demonstrates that the operon is polycistronic. Note that a product spanning each intergenic region was observed ( n =1). d , lva operon induction assay. GFP fluorescence was measured from LB-cultures supplemented with various organic acids (20 mM) ( n =3, biological). Error bars represent s.d. Insert shows the schematic of transcriptional GFP fusion used to test induction of the lva operon. lvaR was cloned onto a plasmid containing its native constitutive promoter and the native promoter region for lvaA . The fluorescent protein sfGFP was cloned in place of lvaA . e , Proposed pathway for LA metabolism. LA, levulinic acid; 4HV, 4-hydroxyvalerate; 3HV, 3-hydroxyvalerate; LA-CoA, levulinyl-CoA; 4HV-CoA, 4-hydroxyvaleryl-CoA; CoA, coenzyme-A; ATP, adenosine triphosphate; 4PV-CoA, 4-phosphovaleryl-CoA; 3KV-CoA, 3-ketovaleryl-CoA; NAD(P)H, Nicotinamide adenine dinucleotide (phosphate) reduced; GFP, green fluorescent protein.
Article Snippet: The Promega GoScript RT PCR kit was used to generate cDNA using 1 μL of a 10 μM gene specific oligo (JMR2 for lvaR and JMR287 for lvaA ) instead of the random oligo mixture.
Techniques: Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Negative Control, Fluorescence, Clone Assay, Plasmid Preparation
