Part:BBa_K3407003

YmdB: a regulator of RNAseIII activity in E. coli.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

YmdB gene in E.coli codes for O-acetyl-ADP-ribose deacetylase, a small protein of 18.8 kDa (UniProt ID: P0A8D6) that catalyses the deacetylation of OAADPr signalling molecule to ADPr [1][S]. Although the purpose of this protein in E.coli remains vastly unstudied, in some experiments it has shown its ability to inhibit E. coli RNAseIII, a member of the RNAseIII superfamily involved in RNA processing. E. coli RNAseIII has shown its catalytic activity upon dimerisation [2][S], in which YmdB interferes by preventing it in vitro. These assays were performed in presence of the Mn2+ (0.4 mM) cation but not Mg2+ [3][S], which was criticised because they do not reflect in vivo conditions [4][S]. In vivo, E.coli keeps Mn2+ concentrations below 40 uM to prevent toxicity even when exposed in a media containing 0.5 mM of Mn2+ [5][S].

On the other hand, studies performing an overexpression of YmdB showed a reduction in biofilm formation and increased susceptibility to apramycin, two pathways known to be mediated by RNAseIII [6][S], further suggesting YmdB is a modulator of its activity. More in-deep research should be performed to elucidate the exact mechanism of RNAseIII inhibition and confirm the reduction in activity is at a substrate level as a consequence of a direct interaction of both proteins in vivo.

Experimental results

• [[File:T--TUDelft--YmdB.png|thumb|none|400px|Figure 1: SDS-PAGE gel showing the overexpression of YmdB. E. coli BL21 (DE3) is the negative control and E. coli BL21 (DE3) pBbA2k_YmdB contains the part BBa_K3407019. MW (Molecular weight marker, #1610363 Bio-Rad), PI (pre-induction), 4h (4 hours after induction), ON (overnight). All the samples used corresponded to the same OD600.]]

As seen in the SDS-PAGE of the total protein content (Figure 1), a band corresponding to the molecular weight of the C-terminal His-tagged YmdB shows that it was successfully overexpressed after induction with 400 mM anhydrotetracycline. No additional bands can be observed in E. coli BL21 (DE3) pBbE8c_mini3 without induction, indicating that there is no leaky expression.