Crystal Structures Of Sensor Domain Of BaeS And Physiological Function Of BaeS/R In Serratia Marcescens

Bacterial cells have dozens of two-component systems(TCS)that sense and respond to specific stimuli and subsequently allow the cells to cope with a variety of environmental conditions.A classical TCS includes a histidine kinase(HK)on the membrane and a response regulator(RR)in the cytoplasm.Generally,upon sensing environmental changes,signaling begins via autophosphorylation of the sensor protein at a conserved histidine residue.The phosphate group is then transferred to an aspartic acid residue in the so-called receiver domain of the corresponding RR.BaeS,a membrane-bound histidine kinase,senses environmental changes and transduces the information to the cell by catalyzing phosphorylation of the transcription factor BaeR.BaeR activates the expression of eight genes,such as mdtABC,tolC,spy,baeSR,acrD.These genes are involved in the envelope stress response,drug resistance,and metal resistance of E.coli.To elucidate the function of BaeS based on the structure,the crystal of MBP-BaeSSD was successfully obtained in this study.After optimization of the initial condition,a 2.8 A dataset was collected.The crystals belong to space group P43212.The structure of MBP-BaeSSD was solved by molecular replacement method using MBP as researching template.To observe the effects of indole on the protein,the structure of MBP-BaeSSD cocrystallized with 2 mM indole was also solved at 2.9 A.The sensor domain of BaeS adopts a mixed ?/?-fold containing a central four-stranded antiparallel ?-sheet flanked by two helices at N terminal and a short ? helix at the C terminal,which includes 2?-4?-1?.Similar to the crystals obtained without indole,the crystal of MBP-BaeSSD cocrystallized with 2 mM indole also belongs to space group P43212 with similar unit cell parameters.And the BaeSSD includes 2?-1?-3?.The first obvious change is happened in the central ?-sheet;the central anti-parallel ?-sheet is composed of three ?-strands in the cocrystallized structure with indole rather than four strands in the non-cocrystallized structure.The angle between the second a helix and the loop which link to ?-sheet of the two structures is also different.The loop between first and second a helix in the BaeSSD cocrystallized with indole is intact while the loop in the native BaeSSD is not observed.Thus,we can conclude that,indole leads to these structure changes of the BaeSSD.But just viewing from the electron density map of the structure,we didnot observe electruon density for the molecule of indole,this may due to the interaction of MBP and BaeSSD may lead to indole could not bind at the protein directly.To investigate the physiological function of BaeS/R,the baeSR,baeR and baeS deletion mutants were constructed.Phenotye analysis showed that the deletion of baeSR increased the resistance in kanamycin,while the single gene mutants did not have any effect on kanamycin resistance.The baeR and baeS deletion mutants were growth inhibited apparently by high concentration of sodium tungstate,while the baeSR mutant grow better than the baeR and baeS mutants,suggesting some contribution to tungstate detoxification but only as an ancillary role to BaeR.BaeR was required for S.marcescens FS14 resistance to sodium tungstate,it is coincidence with the result of BaeR is critically required for tungstate waste disposal in Salmonella Typhimurium reported by Appia-Ayme et al.(Appia-Ayme et al.,2011).