Establishment of a real-time PCR protocol for expression studies of secondary metabolite biosynthetic gene clusters in the G/C-rich myxobacterium Sorangium cellulosum So ce56.
In the attempt to establish a reliable real-time PCR protocol for transcriptional analysis of secondary metabolism in Sorangium cellulosum strain So ce56, a RNA extraction method and a reverse transcription protocol was developed. In order to validate chivosazol or etnangien gene cluster transcripts as good candidates to develop the real-time PCR protocol, stability measurements of the transcripts were performed proving both transcripts to be very stable. The chivosazol biosynthetic gene cluster was taken as the test case to evaluate the special problems arising from the large size of the transcripts and the high G/C-content of the encoding DNA. A set of primer pairs targeting the presumed 90 kbp chivosazol transcript at different positions was employed. The production rate of chivosazol was compared to the transcription of the operon in time course experiments revealing that during the logarithmic growth phase transcription is maximally induced and levels out during the stationary phase. Some deviations in transcript numbers could be measured depending on the primer pair used, but cross-evaluation strengthened the notion that the measured numbers reflect the whole transcript quantities and the in vivo level. Finally, a putative promoter located between chiA and chiB was examined by using the developed real-time PCR protocol.
Kegler
Carsten
C
Pharmaceutical Biotechnology, Saarland University, P.O. Box 151150, D-66041 Saarbrücken, Germany.
Gerth
Klaus
K
Müller
Rolf
R
D020869:Gene Expression Profiling / Q000379:methods; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D008025:Ligases / Q000096:biosynthesis* / Q000235:genetics; D018942:Macrolides / Q000378:metabolism*; D009231:Myxococcales / Q000201:enzymology* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D018942:Macrolides; C096623:chivosazol A; D008025:Ligases