Regulation Of MapZ And Its Phosphorylation On The Cell Division Of Streptococcus Suis

Cell division is the most basic life process in the growth and propagation of bacteria,most of which are carried out in a two-division manner,requiring the participation of multiple proteins.Bacterial cell division protein machinery may become new targets for antibacterial drugs.Their structure and molecular mechanisms regulating cell division have become one of the research hotspots in the field of life sciences,and rapid progress has been made in the study of bacilli.The cell division mechanism of cocci(including ellipsoidea)is different from that of bacilli in many ways.For example,in most bacilli,precise positioning of the cell division machinery(including the Z-ring and diaphragm)at the cell center is controlled by the nucleoid occlusion(NO)and the Min system,which prevent FtsZ from assembling anywhere else other than at midcell.However,two system is missing in cocci.The positioning mechanism of the Z-ring and diaphragm of cocci remains a mystery.In recent years,an anchor protein Z(Mid-cell-anchored protein Z,MapZ)has been discovered in Streptococcus,Lactococcus and Enterococcus.The current research in Streptococcus pneumoniae believes that MapZ is a phosphorylated scaffold protein that arrives early at the cell equator before FtsZ.The MapZ forms ring structures at the division site,as the cell elongates,the MapZ ring splits in two new rings that move apart by the synthesis of peptidoglycan that occurs at mid-cell and that elongates the cell.On completion of cell elongation,and synthesis of the new cell halves,the two MapZ rings are thus positioned at the future division site(the cell equator)of the two daughter cells.MapZ interacts with FtsZ to locate the Z ring and the diaphragm(Fleurie et al.,Nature 2014).Is this new mechanism of positioning Z-ring and septum conserved in cocci? How the different domains and phosphorylation status of MapZ protein affect its function? In this study,the zoonotic Streptococcus suis was used as the research material to explore the regulation mechanism of MapZ on the growth and cell division of Streptococcus suis and the effect of MapZ phosphorylation on its function.1.The regulatory effect of MapZ on the growth and cell division of Streptococcus suisIn order to study the effect of MapZ on the growth and cell division of Streptococcus suis,we have constructed the mapZ-null mutant(?mapZ)and the complementary strain of ?mapZ.The phenotypic comparison showed that ?mapZ displayed impaired growth,its virulence was reduced,its morphology was abnormal,the cells became shorter and rounder,and the cells were stuck together.It looked like a bunch of grapes under the scanning electron microscope.Transmission electron microscopy showed that absence of MapZ results in septum misplacement.In many cells septa were clearly shifted from the center which causes the cells to lose geometric symmetry,generating daughter cells of unequal size.Occasionally,cells with more than one incipient septum were also observedand.Peptidoglycan staining and membrane staining and SIM microscopy observations show that the septum was randomly positioned in ?mapZ cells during the division cycle,the mutant cells looked misshapen,had unequal daughter cell size,and more than one division site appeared in cell,so bacterial cells displayed abnormal chain length and morphology.These results indicate that,similar to Streptococcus pneumoniae,MapZ is involved in guiding septum proper positioning in Streptococcus suis.In order to observe the location of MapZ in Streptococcus suis,we transformed the SC-19 strain with a plasmid expressing GFP-MapZ under the control of the inducible promoter.We can observed that MapZ was located at the equator of the newborn cells;in the early stage of division,MapZ was always located on the septum.As the septum constricts and eventually close,MapZ appears at future division sites.The difference between MapZ positioning in Streptococcus pneumoniae is that when MapZ is positioned on the cell equator,it is first positioned on the outer periphery to form ring structures,and then concentrated on the entire septum.This difference may be due to the fact that the septal division mechanism of S.suis is different from that of S.pneumoniae;MapZ is enriched on the septum of Streptococcus suis,and appeared localized as two rings,at the respective equators of the future daughter cell when the septum contraction signal appears.MapZ is a membrane protein,which is composed of an N-terminal cytoplasmic domain,a single transmembrane(TM)region,and a C-terminal extracellular part.In order to study the functions of its extracellular and cytoplasmic domains,we respectively constructed two domain deletion strains MapZ?extra and MapZ?cyto,both of which exhibit morphological and growth defects similar to ?mapZ,indicating that both the cytoplasmic and the extracellular domains are required for MapZ cellular function.The functions are important.We deleted the intracellular domains in segments and found that the first two subdomains(MapZ-cyto1,MapZ-cyto2)at the amino terminus are important to MapZ,while the third subdomain(MapZ-cyto3)has little effect on the function of MapZ.2.The effect of MapZ protein phosphorylation on its functionThe previous phosphorylation proteomics of our lab found that the MapZ of Streptococcus suis may be phosphorylated by STK.In vitro phosphorylation,MapZ can indeed be phosphorylated by STK,and through point mutation and phosphorylation,we determined MapZ is phosphorylated on Thr26 and Thr66.To investigate phosphorylation of MapZ how affect its function,we constructed 6mutants,including three mutants that encoding the phosphoablative form of MapZ(MapZ-26 E,MapZ-66 E,MapZ-2TE)and three mutants that encoding the phosphomimetic form(MapZ-26 A,MapZ-66 A,MapZ-2TA).Under SIM microscope observation,we found that these two types of point mutants have morphological abnormalities that are not exactly the same as those of ?mapZ strains,which are mainly manifested by asymmetric division of bacteria,expansion or shrinkage of cells,and imbalance of the ratio of length to width of bacteria,but they retained septal localization.It shows that the phosphorylation status of MapZ will affect its function.3.The interaction of MapZ with other cell division proteinsMany cell division proteins perform their functions by interacting to form protein complexes.In order to study which cell division proteins MapZ interacts with to affect its own localization and function,we first screened FtsZ,STK,Gps B,Ezr A and other MapZ interaction proteins with pull-down and bacterial two-hybrid.Using MapZ-cyto1+2,2+3,1+3 and MapZ-extra as bait proteins,bacterial two-hybrid system was used to detect whether the changes in MapZ domains affect the interaction between MapZ and other important cell division proteins.The results showed that MapZ defecting the cytoplasmic domain can no longer interact with STK,Gps B,Ezr A and other proteins,indicating that the cytoplasmic domain of MapZ interacts with these cell division proteins.Using MapZ-2TA and MapZ-2TE as baits,bacterial two-hybridization was used to detect whether phosphorylation pattern of MapZ affect the interaction of MapZ with other important cell division proteins.The results show that there is no significant difference in the interaction strength between MapZ,MapZ-2TA,MapZ-2TE and STK,Gps B,Ezr A and other split proteins.The phosphorylation of MapZ may only affect bacterial cell division in a certain time and space,and this process is difficult to capture by bacterial two-hybrid.We have examined the localization of MapZ,FtsZ,STK,GpsB,MreC,PBP2X and PBP2B in the ?mapZ,and found that these important cell division proteins are often mislocated in the ?mapZ.We further observed the positioning of MapZ and FtsZ in the MapZ-2TA and MapZ-2TE strains,and found that in the normal morphological MapZ-2TA and MapZ-2TE cells,the positioning of MapZ and FtsZ was normal;in some abnormal cells,MapZ and FtsZ are abnormally located,and FtsZ-GFP forms clusters in atypical positions.It further suggests that MapZ phosphorylation may have spatiotemporal regulatory effects.Conversely,we observed the location of MapZ in ?stk,?gpsB and ?ezrA,and found that MapZ failed to position correctly,suggesting that the localization and function of MapZ may be regulated by GpsB and STK.