Extending the classification of bacterial transcription factors beyond the helix-turn-helix motif as an alternative approach to discover new cis/trans relationships

Abstract

Transcription factors (TFs) of bacterial helix-turn-helix superfamilies exhibit different effector-binding domains (EBDs) fused to a DNA-binding domain with a common feature. In a previous study of the GntR superfamily, we demonstrated that classifying members into subfamilies according to the EBD heterogeneity highlighted unsuspected and accurate TF-binding site signatures. In this work, we present how such in silico analysis can provide prediction tools to discover new cis/trans relationships. The TF-binding site consensus of the HutC/GntR subfamily was used to (i) predict target sites within the Streptomyces coelicolor genome, (ii) discover a new HutC/GntR regulon and (iii) discover its specific TF. By scanning the S.coelicolor genome we identified a presumed new HutC regulon that comprises genes of the phosphotransferase system (PTS) specific for the uptake of N-acetylglucosamine (PTS(Nag)). A weight matrix was derived from the compilation of the predicted cis-acting elements upstream of each gene of the presumed regulon. Under the assumption that TFs are often subject to autoregulation, we used this matrix to scan the upstream region of the 24 HutC-like members of S.coelicolor. orf SCO5231 (dasR) was selected as the best candidate according to the high score of a 16 bp sequence identified in its upstream region. Our prediction that DasR regulates the PTS(Nag) regulon was confirmed by in vivo and in vitro experiments. In conclusion, our in silico approach permitted to highlight the specific TF of a regulon out of the 673 orfs annotated as 'regulatory proteins' within the genome of S.coelicolor.

Figure 1

Overall strategy for the prediction of HutC/GntR binding sites in a genome of interest (gray) and for the selection the best HutC/GntR candidate to regulate genes with a specific HutC-like cis element pattern (black). For further explanation see text.

Figure 2

The N-acetylglucosamine uptake in S.coelicolor. (A) Individual loci of genes of the PTS^Nag regulon. Black boxes indicate positions of the predicted HutC/GntR binding sites. (B) Model of N-acetylglucosamine uptake via the PTS in S.coelicolor. For further explanations see text in the result section.

Figure 3

Alignment and weight matrix deduced for the PTS^Nag cluster.

Figure 4

In vitro and in vivo experimental validation of DasR/PTS^Nag cis/trans relationship. (A) Electrophoretic mobility shift assays. A single HutC/GntR-binding site was present within probes I and II while two were identified in probes III and IV. Probe V, containing the predicted cis-element of CRP of S.coelicolor, was used as control. Probe VI contains the predicted cis-element upstream dasR that presented the best score against the ‘PTScoeli’ weight matrix (see Table 1). A DasR/DNA complex formation was observed for all tested HutC-like cis sites (probes I to IV). (B) In trans effect of DasR on N-acetylglucosamine transport. Mycelia of M145 (pUWL-SK+), the control, and M145 (pFT241 dasR⁺), the DasR overproducing strain, were grown on 50 mM glycerol in the presence and absence of 50 mM N-acetylglucosamine. Data points to determine the initial uptake rates were collected as triplicates and the experiment was reproduced three times. Error bars indicate the derived standard deviations.