Development of a Corynebacterium glutamicum DNA microarray and validation by genome-wide expression profiling during growth with propionate as carbon source.
A DNA microarray was developed to analyse global gene expression of the amino acid-producing bacterium Corynebacterium glutamicum. PCR products representing 93.4% of the predicted C. glutamicum genes were prepared and spotted in quadruplicate onto 3-aminopropyltrimethoxysilane-coated glass slides. The applicability of the C. glutamicum DNA microarray was demonstrated by co-hybridisation with fluorescently labelled cDNA probes. Analysis of the technical variance revealed that C. glutamicum genes detected with different intensities resulting in ratios greater than 1.52 or smaller than -1.52 can be regarded as differentially expressed with a confidence level of greater than 95%. In a validation example, we measured changes of the mRNA levels during growth of C. glutamicum with acetate and propionate as carbon sources. Acetate-grown C. glutamicum cultures were used as reference. At the 95% confidence interval, 117 genes revealed increased transcript levels in the presence of propionate, while 43 genes showed a decreased expression compared with the acetate-grown culture. Global expression profiling confirmed the induction of the prpD2B2C2 gene cluster already known to be essential for propionate degradation via the 2-methylcitrate cycle. Besides many genes of unknown function, the paralogous prpD1B1C1 gene cluster as well as fasI-B (encoding fatty-acid synthase IB), dtsR1 and dtsR2 (components of acyl-CoA carboxylases), gluABCD (glutamate transport system), putP (proline transport system), and pyc (pyruvate carboxylase) showed significantly increased expression levels. Differential expression of these genes was confirmed by real-time reverse transcription (RT) PCR assays.
Hüser
Andrea T
AT
Lehrstuhl für Genetik, Universität Bielefeld, Universitätsstrasse 25, Bielefeld D-33615, Germany.
Becker
Anke
A
Brune
Iris
I
Dondrup
Michael
M
Kalinowski
Jörn
J
Plassmeier
Jens
J
Pühler
Alfred
A
Wiegräbe
Iris
I
Tauch
Andreas
A
D002244:Carbon / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D002874:Chromosome Mapping / Q000295:instrumentation* / Q000379:methods; D003352:Corynebacterium / Q000166:cytology / Q000502:physiology*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020869:Gene Expression Profiling / Q000295:instrumentation* / Q000379:methods; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D016680:Genome, Bacterial; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D011422:Propionates / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D013045:Species Specificity
D011422:Propionates; D002244:Carbon