Abstract
The synthesis of a variety of proteins, including the well characterized degradative enzymes, which occurs during the transition state between vegetative growth and the onset of sporulation in Bacillus subtilis is controlled by a class of molecules known as transition state regulators. One of these regulators is the product of the hpr gene, first identified by mutations affecting the synthesis of extracellular proteases. We have purified the Hpr protein and found that it binds specifically to DNA fragments carrying the promoters and the upstream regions of the alkaline (aprE) and neutral (nprE) protease genes of B. subtilis. DNase I protection experiments revealed that the Hpr protein is able to bind at four and two regions of the aprE and nprE promoters, respectively. We have also located two Hpr binding sites in the promoter region of a gene of unknown function which is nevertheless known to be developmentally regulated during the transition state and which occurs in the same operon as the gene encoding another transition state regulator, Sin. The location of one of the Hpr binding sites on the aprE gene occurs adjacent to a region to which the Sin protein binds. However, in mixing competition experiments we have shown that Hpr and Sin binding occurred independently, and no visible alterations of protected regions were detected.
Highlights
Developmentally regulated during the transitionstate The hpr gene codes for a protein (Hpr) of approximately and which occurs in the same operon as the gene en- 23,700 Da (Perego and Hoch, 1988) which is involved in the coding another transitionstate regulator, Sin
In mixing competition experiments mutants andinsertional inactivation experiments have shown we haveshownthat Hprand Sin bindingoccurred that Hpr is a negative regulator of these proteases (Perego independently, and novisible alterationsof protected and Hoch, 1988)
Expression of the Hpr Protein inE. coli-Our strategy was to clone the hpr gene ontoan manipulated E. coli expression vector to produce sufficient amounts of the Hpr protein to serve as thesource for its purification
Summary
The dialyzed fraction was reloaded onto an 8 X 3-cm heparinwtapcErnaearfDhriloclJeteceo.ceNeecMn,noBcBDrmchoeAcRtAa1taa1hNiarornlp9cIacdGoi5pr-t,Afu8tnti,picH1erre.nebr9iogoorrcnGg(iinaIiimeTrnoThlnaag)danridteta,lcotaAoieDawc1s.Eitcmn,loShnhe1wst.Hed,ndnedth1aSrracihui(e5efaeqfosso1sts(rriatuighl0uEaiolndpnineir0gitmnRsrgwososghmo,aip1-awpSpar(altEBgt7iP.eoleGasen,cFa./(cnerclndmeseoaoi-troto1mrshnsnll-l9cn,owoimeei~h8flaYFsdo(iLai6e,mdtppfdr8th,B)mwaaeJSsp0BEM'Mnd,c-aiDimdR~cnaePccsui2airt)H,o)ileueshsrln4nas,Rswlcldylie5an9idunseoiinsaImaa,7eaunpg,d-scrCaImgEHoeac(tcCpt4ZorduaPhiheRtp4bAnlAlse1lhetpy,ealutdirLM0)seasvP)rXep.bIi.aae1pa-gInpTnowbatITtg,5olpeuii-Kroeel/tohdAccrsiemmrrhnn6Ehue(aet(igBlf.tetaL1ro-CnpBtloikgTmaru.rlotKmo,.aieabe,h-ecennn1nQdoRgssetZdea9sEru.hyVETawiamf8Ybor.tdwot.i4h8AtiAdooyF)rit)cae,lr.mh-nc(eoicsiorSs1sie~rloeafn-eir-storEndpPak)rpplHbe.atprgsilAllatlaiaaicuorlaoFotissshoclchin()brmmmm)nepGeheia-eUbnlririieselddiddygleses--t69pw5paaaa6oBlfoKu(,argGd0omrsvsniaaCboaid0oomedCscantoirtllrethentu/eogeoerlecimdOseoaidorcsxnocnMatweh,Anpstronaneotwanie.rarcrccbetnxaiyusAoTaeibecKs2lpddcsln.sdusBh0u,Crtloi,atsdfioe%oi1mb1rfofn1tsisaFnaeoya9na4sdnrTtrlfgr8fli7e.Lye,oaryeioor6.0dandzfnwdoasnTfA)caeidcHw,ptuavashtldhoaeiiKsseurPteeone)cnihy(cg-nnrnSoodH2t2etoclteofgSo0Indi0.opttw-r0i,oceCplarn(aTu"aeiuaumlSenCpms.nphnrrMnTcpi2ri(K.tedncSfntro1rr1hrHiioPioQaet9.eiKpett5enptqteirdp5hVpeaiiCocrqu1nroesilseeXt4puneI)iHnnepn)anniCriai.-lntpptoeacgnwni13rltTrblretsiaeed0-Euonaerrhtncmactsvpaiei.Lmeeebc(notderccpAdeaopondoioftdyavsrtipcslaDnueneinneupep.scsisaE.pednnlnAftiaeeliTanrorAster(nnesabcrguy1RthdwvtcEtaloci9roieoetietavBa-cati5oiifsnlTopnseeotd7uelhieoanrs,iorna)sapnosirtts.swalrssstefwhshetyAcywnaueraaeehbwsefc(idlmsorwtoneArreef-eeeyriFig21lgirmmnmnnnpotldeMl5.oeh5yotwresi0enuosiecc-)tddr-eum-nte.bp2eoemeetgrrd0tahnoogtromaig0lerarm)pp.n(ilaoe,ct2InieeTwtisiaBnutm5rriiotnetnnooogehh0MaeFndadgnnnhds-deel,)-. Parallel cultures were grown and induced as described and discussed under "Results," and the Hpr proteinwas purified as follows (Fig. 1).Cells were harvested by centrifugation, washed twice with OA buffer (25 mM Tris, pH 8.0, 10 mM KCI, 0.1 and used it to 1)replace the ribosome binding site with one known to function well in E.coli; 2) change the initiatorcodon from TTG to ATG; 3) optimize the spacing and sequence between the ribosome binding site and ATG; and 4) separate the sin gene from the first open reading frame (ORF1) of the operon and place it directly under the control of the tac promoter of pKQV4. With 6 volumes ofOA buffer and the proteins eluted using stepwise Sin Purification-Eight liters of LB-brothinoculated with an increases (2 column volumes each) in the KC1 concentration (50,100, 250, 500, 1,000 mM) The proteins in these fractions were visualized. Specific details concerning these clones are available upon request from the authors
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