Characterization Of Secreted Protease And Regulatory Mechanism In Stenotrophomonas Maltophilia FF11 In Response To Low Temperature

In recent twenty years,as one of increasing food spoilage bacteria and fish pathogens,Stenotrophomonas maltophilia has become a potential threat to aquiculture industry.A major factor is the outstanding ability of S.maltophilia to secrete protease at low temperature.However;the type and the personality of the low-temperature induced protease was not reported in detail.Furthermore,the regulation mechanism about low temperature inducing the protease production in S.maltophilia was not clear.For the above two questions,the study was separated into two parts:one is purification and characterization of the low-temperature induced protease and analysis of the characteristics of the protease and its primary structure in detail.the other was the exploration of the regulation mechanism that how the strain produce the protease in response to temperature.(1)According to enrichment culture method,one strain secreting higher level of protease at low temperature was isolated from frozen Antarctic krill.Based on the analysis of 16S rDNA and biochemical-physiological characteristics,the strain was identified as S.maltophilia,named S.maltophilia FF11.The strain has a higher level of extracellular protease activity at low temperature(15? and 25?),and enzyme activity of culture supernatant at 37? was extremely low.According to zymogram analysis,the major protease was largely produced at low temperature,but not at 37?,and the protease was named SmtP(Stenotrophomonas maltophilia temperature-response protease).According to ammonium sulfate precipitation,Q-Sepharose Fast Flow chromatography and Superdex 75 chromatography,SmtP was purified to homogeneity.SmtP remains high active and stable at high salt concentration(4 M)and in different organic solvents(50%,v/v).Based the protein fingerprint analysis and gene cloning and sequencing,the full-length gene sequence of smtP was obtained(GeneBank with accession number KP399635).The ORF contain 1854 bp,encoding a precursor of 617 amino acids residues.N-terminal sequences(LVPND)and molecular mass of SmtP(37.4 kDa)showed that the mature protease contains 368 amino acids residues,therefore,the primary sequence of SmtP was analyzed in detail.(2)The analysis of quantitative RT-PCR(qRT-PCR)and promoter fusion revealed that temperature influenced the expression of smtP partly at the transcription level.A cAMP receptor protein-like Clp was deleted using sucide vector pEX18Tc by biparental conjugation in S.maltophilia FF11,generating clp deletion strain Aclp.The protease activity was reduced significantly at 15?and 25? in clp mutant strain.Zymogram analysis revealed that the ?clp was not able to produce SmtP any more at low temperature,and the analysis of qRT-PCR and promoter activity indicated that Clp could regulate positively smtP expression in S.maltophilia FF11.Meanwhile,clp expression was also in a temperature-dependent manner.(3)Bioinformatics analysis revealed four putative two-component systems Sm3706/Sm3705,Sm1829/Sm1828,Sm1911/Sm1912 and Sm0737/Sm0738 may being related to intracellular second messenger c-di-GMP metabolism in S.maltophilia.Deletion of four responsive regulators revealed Sm3705 reduced 78-82%the protease activity in S.maltophilia FF11 at low temperature,Sm1828 reduced 50-53%protease activity,Sm1912 reduced 10-15%and Sm0737 had no effect on the protease activity.According to the zymogram analysis,deletion of Sm3705 abolished completely the production of SmtP,a similar result with Aclp,and deletion of Sm1828 reduced obviously the SmtP production.Sm3706/Sm3705 was a novel uncharacterized TCS and could respond to low temperature stimuli,therefore,Sm3706/Sm3705 was named as LotS/LotR(low temperature regulator and sensor).LotS is an unorthodox sensor kinase and LotR is a response regulator that contains GGDEF and EAL domain related c-di-GMP metabolism and REC domain.Both LotS/LotR and Clp mediated smtP expression,suggesting LotS,LotR and Clp are all part of the same regulatory pathway in mediating protease expression.The analysis of Clp promoter activity in AlotS or AlotR and the protease activity analysis after overexpressed Clp in AlotS or AlotR indicated that Clp was a downstream regulator protein of LotS/LotR.The activity of recombinant expressed LotR in vitro revealed that the LotR was unable to synthesize or degrade c-di-GMP.ITC results showed that Clp from S.maltophilia FF11 has an nearly equal affinity ability to c-di-GMP with Kd = 20.3 ± 0.9 nM at 25? and Kd = 22.7 ±1.1 nM at 37?.Therefore,these findings indicated that Clp from S.maltophilia FF11 could bind to c-di-GMP,and temperature could not play a dominant role on the binding ability to c-di-GMP.The level of c-di-GMP was increased in S maltophilia FF11 growing at high temperature,Whereas,the level of c-di-GMP are not in an obvious temperature-dependent manner in AlotR strains,these results indicated that LotR took part in the level of c-di-GMP controlled by temperature.(4)Bioinformatics analysis revealed a high level of sequence identity between the protein sequences of Sm1828/Sm1829 and their homologues RpfC/RpfG in Xanthomonas.sp.?rpfF,?rpfC.?rpfG and their respective complementary strains were constructed,proving that RpfC/RpfG in S.maltophilia FF11 could regulate the extracellular protease activity in response to DSF molecule.Deletion of rpfC or rpfG have no effect on the growth,however,reduced significantly the protease activity in S.maltophilia FF11 at low temperature.According to the zymogram analysis,the production of SmtP was significantly reduced in rpfC and rpfG mutant strains,indicating RpfC/RpfG could regulate protease production in response to temperature changes.The protease activity was significantly reduced in DSF-deficient strain and addition of exogenous DSF to or overexpression of RpfF in wild-type and rpfF mutant cultured at 37?could not increase protease activity.This result indicated that DSF was effective for protease production only at low temperature.The analysis of RpfG enzymatic activity indicated that the activity of RpfG in S.maltophilia FF11 might be dependent on both the low growth temperature and presence of DSF.Above all,the main characteristics of SmtP produced by S.maltophilia FF11 were studied in detail,and the primary sequence of SmtP was analyzed in this study.The transcription factor Clp and the upstream regulatory factor LotS/LotR involving in regulating smtP expression was found.And the preliminary mechanism in response to temperature stimuli by signal transduction system LotS/LotR/Clp was studied.The RpfC/RpfG two-component system in response to quorum sensing signal molecule DSF and temperature stimuli,mediating protease production was studied in S.maltophilia FF11.