Interference of PrfA with glucose uptake

Listeria monocytogenes (Lm) strains expressing high levels of the virulence regulator PrfA (mutant PrfA* or wild-type PrfA) show strong growth inhibition in minimal media when supplemented with glucose but not when supplemented with glucose-6-phosphate compared to the isogenic strains expressing low levels of PrfA. A significantly reduced rate of glucose uptake is observed in the PrfA* over-expressing strain growing in LB supplemented with glucose. Comparative transcriptome analyses were performed with RNA isolated from a prfA mutant and the isogenic strain carrying multiple prfA or prfA* copies on a plasmid. The analysis reveals in addition to high transcriptional up-regulation of the known PrfA-regulated virulence genes (group I), less pronounced up-regulation in the expression of several phage- and metabolic genes (group II) and strong down-regulation of several genes involved mainly in carbon- and nitrogen- metabolism in the PrfA* over-expressing strain (group III). Among the latter genes are in particular the nrgAB-, gltAB-, glnRA- (involved in nitrogen metabolism), ilvB- operons (involved in biosynthesis of the branched chain amino acids) and genes for some ABC transporters. Most of the down-regulated genes have been shown previously in Bacillus subtilis to belong to a class of genes whose expression is negatively affected by impaired glucose uptake. The results lead to the conclusion that excess PrfA(*) interferes with component(s) essential for PTS-mediated glucose transport. Keywords: Metabolism and Virulence gene expression Transcriptome analyses were performed using whole genome DNA microarrays that contain synthetic 70mer oligodeoxyribonucleotides covering all ORFs of the Lm genome. Three independent RNA preparations from the conditions to be tested were pooled and 6 equal aliquots (15-20 µg) of total RNA from the strains were used to synthesize cDNA differentially labelled with Cy3-dCTP and Cy5-dCTP (Amersham Pharmacia, Freiburg, Germany) during a first-strand reverse transcription reaction with Superscript II RNase H- RT and 9 µg random primers (Life Technologies, Karlsruhe, Germany). Dye swap was performed as follows: Three cDNA samples from the one strain were generated using Cy3-dCTP and the other three using Cy5-dCTP and similarly for the other strain to be investigated. The two differentially labelled cDNA samples were combined, diluted to 3 × SSC, 0.1% (wt/vol) sodium dodecyl sulfate, hybridized to the microarray slide and incubated at 65°C for 16 hrs. Six such slides were hybridized using the generated probes for each strain combination. In this study there were 3 strain combinations in all.