| Reversible phosphorylation,which plays a crucial regulatory role in prokaryotes and eukaryotes.Protein phosphorylation are mainly catalyzed by histidine kinase,serine/threonine kinase and tyrosine kinase in bacteria,while the functions of histidine kinase of two-component system have been extensively investigated.In recent years,eukaryotic-like serine/threonine kinase（STK）and phosphatase（STP）have attracted much attention in bacteria.However,there are multiple STK in most bacteria,which leaded to functional redundancy.It is difficult to analyze the regulatory function of each STK.We found that mostly Streptococcus have only one paire of STK/STP,which played an excellent material for studying the regulatory mechanism of STK and STP.Many studies used Streptococcus suis（S.suis）as material to study the regulatory function of STK or STP.It was found that STK and STP play an important role in regulating S.suis growth,metabolism,and pathogenicity.The regulatory effects showed opposite or consistent,while the regulatory mechanism was not clear.As a paire of phosphorylation/dephosphorylation system,STK/STP has parallel or cross signal regulatory networks,which regulated many different physiological and pathogenic processes and could not be studied in isolation.In this study,we first compared the regulation of STK and STP on the growth,cell division,and virulence of S.suis,then analyzed the regulation of STK/STP on the protein expression and protein phosphorylation of S.suis by proteomic and phosphoproteomics,and partially explained the mechanism of STK/STP regulating on the growth,cell division and pathogenicity of S.suis.This study also revealed the different localization patterns of STK in S.suis and Streptococcus pneumoniae（S.pneumoniae）and the important role of PASTA domain of STK in localization.Then constructed the interaction network between STK and cell division-related proteins.This study laid a foundation for insight into the mechanism of STK regulating on cell division of Streptococcus.1.Regulating of STK and STP on the growth and virulence of S.suisIn this study,S.suis type 2 SC-19 strain was used as the study material,the single gene deletion strains of stk or stp gene（Δstk,Δstp）and double genes deletion strains（ΔstpΔstk）were constructed,and systematically comparative the effects of STK or/and STP deletion on the growth,morphology and pathogenicity of S.suis.In nutrient-rich TSB medium,the growth curve ofΔstp andΔstpΔstk were similar to that of SC-19,while the OD600 ofΔstk reached a lower value than that of SC-19 in the plateau stage.In the chemically defined minimum medium（CDM）,Δstk could hardly grow,the growth ofΔstp was not affected,and the growth rate ofΔstpΔstk decreased,but faster than that ofΔstk.The growth defect ofΔstk was significantly restored by adding complex nutrients（tryptone or peptone）and multivitamins to CDM medium.The results showed that under the condition of nutrient-rich medium,the growth ofΔstk was not affected,but under the condition of minimum nutrition,the growth ofΔstk was seriously impaired,and vitamins could significantly recover the growth defect ofΔstk.In addition,Δstp,Δstk,andΔstpΔstk formed long chains.It could be seen that both STK and STP regulated the morphology,growth and metabolism of S.suis,and STK plays a more significant role than STP.Further morphological analysis found thatΔstp formed round cells and the angle between Z-ring and cell long axis was abnormal,whileΔstk formed multiple Z-rings with uncontraction,and the cell morphology ofΔstpΔstk was relatively normal.The statistics of the length and width ratio of cells showed thatΔstp,Δstk,andΔstpΔstk significantly decreased.In order to further understand the regulatory effects of STK and STP on cell division of S.suis,the phosphatase activities ofΔstp andΔstk were determined.The results showed that the total phosphatase activity ofΔstp significantly decrease,while that ofΔstk had no significant change.The results suggested that both STK and STP play an important role in regulating S.suis cell division.In order to compare the regulatory effects of STK and STP on the pathogenicity of S.suis,a series of pathogenicity assays in vitro and in vivo were used to evaluate the characteristics of SC-19,Δstk,Δstp andΔstpΔstk.Biofilm formation assay showed that the biofilm forming ability ofΔstk andΔstpΔstk were significantly enhanced,whileΔstp had no significant change.Mouse macrophage phagocytosis assay showed that the anti-phagocytic ability ofΔstp,Δstk andΔstpΔstk significantly decreased.The pathogenicity assay showed that the mortality of mice infected withΔstk andΔstpΔstk were significantly lower than that of SC-19,while no significantly changed inΔstp.Colonization assay showed that the colonization ability ofΔstp,Δstk andΔstpΔstk significantly decreased.The competitive colonization assay showed that the colonization ability ofΔstk was weaker than that ofΔstp,whileΔstpΔstk was between the two.To sum up,it could be seen that STK plays a more important role in pathogenicity regulation than STP.2.Regulation of protein expression and global phosphorylation by STK and STP in S.suisIn this study,the global protein expression and phosphorylation ofΔstk,Δstp and SC-19 strains have been revealed by proteomic and phosphoproteomics,which partly explained the mechanism of STK/STP regulating on the growth,cell division and pathogenicity of S.suis.TMT proteomic analysis showed that there were 139 differentially expressed proteins inΔstk,including 37 up-regulated and 102 down-regulated,while 82 differentially expressed proteins inΔstp showed 47 up-regulated and 35 down-regulated.The results showed that stk deletion had a greater effect on the global protein expression of S.suis,which was consistent with its more significant effect on phenotype.InΔstk,the differentially expressed proteins most frequently fell in categories of inorganic ion metabolism,transcription and nucleotide metabolism,while fell in categories of amino acid metabolism,inorganic ion metabolism and energy metabolism inΔstp.Differentially expressed analysis of metabolism-related proteins showed that Co A binding protein and pyridoxine kinase were significantly down-regulated inΔstk,while no significant change inΔstp,suggesting that STK plays an important role in vitamin metabolism of S.suis.Differentially expressed analysis of virulence-related factors（VFs）showed that more VFs showed down-regulated in the?stk than in the?stp,including Ssn A,DPS and Ide S were significantly down-regulated in?stk,while no significant change in?stp.In summary,it could be seen that STK plays a more important role in the regulation on the growth,metabolism,and pathogenicity than STP.The phosphorylated proteins ofΔstk,Δstp and SC-19 strains were enriched by serine/threonine phosphorylation antibody and analyzed by mass spectrometry.Compared with SC-19 strain,there were 67 differentially phosphorylated proteins（39 proteins up-regulated and 29 proteins down-regulated）inΔstk,while 14 proteins up-regulated and 38proteins down-regulated in 50 differentially phosphorylated proteins inΔstp.InΔstk,1/3of the proteins with reduced phosphorylation level were related to cell division,including Gps B,Map Z,Fts Z,Sep F,Div IVA,Fts W,Mlt G,Jag,and Glm S.However,Map Z,Gps B,Fts Z,Div IVA,and Jag have been confirmed to be the substrates of STK,while other proteins were mainly related to metabolism and protein translation.Among the proteins with increased phosphorylation level inΔstp,four proteins were related to cell division,including Div IVA,STK,Mlt G and Glm M,while other proteins were mainly related to metabolism.It could be seen that STK and STP play an important role in cell division of S.suis.The mechanism of protein phosphorylation in regulating cell division worthing further study.3.The mechanism of STK regulating on cell division of StreptococcusPrevious and present studies have suggested that STK（a transmembrane protein）was a component of Streptococcus cell division complex,and whether it was directly（not just through phosphorylation modification）involved in the process of Streptococcus cell division was unclear.In this study,the subcellular localization patterns of STK protein in S.suis and Streptococcus pneumoniae（S.pneumoniae）were compared,and the interaction network between STK and cell division-related proteins was constructed,which laid a foundation for further exploring the mechanism of STK regulating on Streptococcus cell division.The subcellular localization of STKSS（STK protein of S.suis）showed that located in division site at the early stage,then moved with the movement of the cell division site,while the STK protein of S.pneumoniae(STKSP)was always located in division site and did not move with the movement of the division site.When the expression plasmid of STKSP was transformed into S.suis,it was found that the localization pattern of STKSPin S.suis was the same as that of STKSS,which may be due to the formation of heterodimer between STKSP and STKSS in S.suis.Bioinformatics analysis showed that the amino acid sequences of STKSS and STKSP in kinase domains were highly similar,however the similarity of their extracellular PASTA domains was low.The PASTA domain of STKSSwas replaced with the PASTA domain of STKSP to form the hybrid STKSS(PASTASP).The localization of the hybrid in S.suis was similar to that of STKSP in S.pneumoniae.It could be seen that the PASTA domain was the key factor determining the localization pattern of STK in the cell division process of Streptococcus.Bacterial two hybrid experiment found that STK has a wide range of interactions with division-related proteins,including Mru A1,Mru C,Mru Z,Mru D,Mru G,Mra Y,Mlt G,Fts W,Fts E,Fts X,Fts Q,Fts L,Fts B,Mre C,Mre D,PBP2x,PBP2a,PBP1,PBP2b,Fts Z,Fts A,Div IVA,Sep F,Map Z,Ezr A,and Wih A.Only some of these proteins have been confirmed to be substrates of STK,suggesting that STK may also regulate cell division through interactions. |
