Bacillus subtilis two component system YvfT-YvfU is involved in thermal regulation of the ABC transporter YvfRS

FERNÁNDEZ, P; PORRINI, L; ALBANESI, D; DE MENDOZA, D; MANSILLA, MC

Rosario

Congreso; IX CONGRESO ARGENTINO DE MICROBIOLOGIA GENERAL SAMIGE 2013; 2013

Bacteria can encounter a wide range of environments and must adapt to new conditions in order to survive. As the selective barrier between living cells and their environment, the plasma membrane plays a key role in cell viability. The barrier function of the cytoplasmic membrane is known to depend critically on the physical state of lipid bilayers, making it susceptible to changes in environmental temperature. For signal transduction across the cell membrane, bacteria extensively use two-component systems (TCS), which have an input-sensing domain (histidine kinase, HK) and an output effector domain (response regulator, RR). The Des circuit of the Gram-positive bacteria Bacillus subtilis regulates the expression at low temperature of the fatty acid desaturase encoded by the des gene. It is composed by an autophosphorylatable HK (DesK), and a DNA binding RR (DesR). It has recently been demostrated that overexpression of DesR in B.subtilis desK null mutants results not only in the induction of the des gene but also of an operon formed by the genes yvfRSTU, which encodes a putative ABC transporter and a two component system. The bioinformatic analysis of YvfT-YvfU sequence showed a high identity with DesK-DesR. In adittion, the inverted repeated boxes in the promoter region of yvfR are very similar to that of the regulatory region of the des promoter. In this work, we investigated whether YvfTU mediates activation of the ABC transporter YvfRS. We performed β-galactosidase assays of B.subtilis yvfTU null mutants carrying a transcriptional fusion of PyvfR to lacZ (CM49). We found that transcription from PyvfR is activated only if YvfTU is expressed in trans, indicating that this TCS is essential for regulation of the transporter expression. Besides, such expression occurs at 37°C and not at 25°C. We also observed that YvfTU expression from an inducible promoter recovers the ability of a DesKR¯ mutant to express the des gene at low temperature, which implies an opposite behavior to the one seen with PyvfR. We also found that transcription of yvfR in CM49 is also activated when the regulator YvfU is expressed in trans, but not by YvfT alone. Surprisingly, this expression depends on the presence of DesR. To know whether YvfU induces yvfR operon in its unphosphorylated form or if it ought to be phosphorylated by DesK, we constructed a B. subtilis yvfTU null mutant that also is DesK¯, carrying the transcriptional fusion PyvfR-lacZ. We can conclude that the ABC transporter YvfRS is regulated by the TCS YvfTU, in a thermal-dependent fashion (activated at 37°C and not at 25°C). For the transcriptional activation of the yvfR gene, an interaction between both systems, DesKR and YvfTU is required. The different behavior of YvfR on Pdes and PyvfR might be explained by slight differences between the sequences of regulator-binding regions in each promoter.