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The effect of histone on the inducible β-galactosidase in Escherichia coli
1. 1NTRODUCTION Extensive evidence hae been preeented in a%?cent years that bask protei”8, in particuhr histo”e. hwe B” imaortant renetic rea!Iatorv inction’i” the c& oi higher xgan~sms (ior detailed intormatio” see Busch, 19S5). The question, whether oilla cl lower organisms contain histone or not, eeems to have been solved recently. Tontno and Ftoeijn (19S6) found DNA-assodlted histone in baker’s yeast, and Leaver and Crolts (1966) observed that rlbonome al different bacteria contnined hiBto”e: however, only IittIe If any of thelr DNA was in ibe form of a typical “ucleohistone complen. The function of histone l” the @Ia of lower organism” is not clear ““d data from thin field are only acant. The present study was undertake” to establIg the effect hi&one on some aspects of protein synthesis in E. coli. Inducible P-galnctobidaae of .E. coli MI, 30 has UBen chosen as D model protein.
of
Htstone was added to the growing culture after two cell dlvIaIo”s: the tnducer, ‘I’MO(lO-3b%), was added ten minrtee Iater. After a suItable time LntervaI aamoles were taken and treated with cbIoramphe&oI (109 pg/ml) and t&t&e. The aeszy of P-gaLactoaldase In the sunples followed in Ledertmg’a procedure (1950), and wan performed as dee.crUwd prerIoosly (mefI”oVP et al., 1865). Our results are expressed I” te:r”s of epecu*c activity (mp moIes or o-nitrophe”o1 llbelwed from ON% per minute per mg dry weight of the cuIt”re). The percentage of lnhlbltlo” of enzyme i’wmatlon b] histone was cirleulated with regard lo thc speclflc activity of control ceIIn which were “ot treated rith hlr4one. Histone-cbioride ww prepvca from CaIf thymus ad kindly su~lted ky Dr. K. R?Blra Jr. @errnIne tetrahydrc&Iorlde wan purchased from Calblochem. The &r&e were I( glfl of Dr. II. V. Rlcke”berg.
general
3. RESULTS 2. MATERIALS
AND METHODS
The foIIowlng &rains were wed:E.co!l ML 30 l”duclBIe for P-g;lectosldase, andE.cofi ML 308 CO”etlhitive for 8-galaetosldaee, Tho bacteria were inoculated from an overnight culture, and grow” in P reoiprocat‘ng wo&r-bath shaker at 3’ioC in the mineral medium M 56 (Monad et al., 1951) with 5 X 10-3 M II&tow as the sole ~o”rce of carbon. Changes of ooace”tra:lo” of MS+’ and Cat+ WI be indicated In the results.
AND DISCUSSION
It foIlows from the .?~~rime”t preSented I” table 1 that hlsto”e inblbltn the growth of E. coti culture and the i”&clb1e eynthe*ls of #?-galactosldaee. Thla effect depends upon ‘%e cr~neentratie” of Mk ions prewnt in the gro*lh medium. Higher co”ce”tral1oM of I@++ prewlted lhle in. hlbltory actlo” cd hlsto”e, hut the l”h0ltbn of enrymc coiace”!raIo”B of hlstcae used I” our experImco:s (50.409 &I”l) are very hfgh lo comparlsc% with the ctmcentratfcm of 1 &ml whlct wap. deal@&d by IIlrach
famatimM morepronounced thanthe t”htbitt”nof growth.
(1956) aa MuI for E.coli cells. However, HIreoh suepaled the celle In e cltrete-phospbate lmffer end found, in accordance wlth WP reeults, that P higher ccacer.trotlw of poaltlwly charged lone (0.15 - 0.2 M NnCl) blocked the btbal ectlon cd hlstom?. In the concentrrt!nn rage from 5 X 10-4 M to 7.5 x lo-* H Mg+‘, and 1O-4 M CR++ (which te the ueuel cmceatratton of Ca++ ln the mineral
medium M 55) histonc at a concentration of 400 pg/ml prcfereritialty lnhiblted P-galactasidase syntheale without pronounced inhibition of growth. On the basis of this observation we attempted to matntatn in the f~UowIng expeFimente an optimal ratlo of histone: Mg++, which would exert the most pronounced effect on the activity of Inducible ,%galacto,sidase Without influencing the growth of the cultwe. One of these experlmanta IS presented in fig. 1. It can be seen that hletone lnhlbited the Inducible synthesis of P-galactostdase during the logarithmic phase of growth. In the stationary phase, cells treated sltb hlstone reached the fame speclflc activity of enzyme as the control and, mo~over, a stimulatlon of enzyme syrrthesls over the control level was observed In some earlier communications Srow our laboratory (Burger and Pavlas0va, 1954: NeEinov% et al., 1965; NeClnnv6 and Burger, 1965: Lefk&t8 and Burger, 1805) we reported that both in uflro or in uiuo, histone cculd liberate /J-&Plectosidase and amylomalhse bound to cell etructu.es in come E. coli mutants. Hence we cmld the posslbllity that the histone Mlbltion of s-g.llnctostdaseformatlon wee not due to an achtal Inhibitton of its eydhesis. We postu:ated the alternattve explana” m that histone in
notexclude
the logarithmic phsse caused S btndtng of the enzyme to Cell structures and tts cansequent bratttvatlon. 0” the other hand, In the StStionary phase, P-gaiaofostdase ~1s liberated from these structures by the htstone present. This pcssibiltty was rules out by the following SVidellCe: a) In thS presence of cblorSmphenlco1 added to the induced culture at the beginning of the stattonary ,,hnse of growth, no further enzyme pro&ction to.& place or, bl‘2tiler wordS, “Cl eneyme was ‘liberated’ by histone in the stationary phase.
b) The ~ratucttcn af constitutive @-gglactoSid1se In the ML 308 BtraIn wSS not irdluenced by bistone either tn the logSrtthmte or in the Stationary phase. c) p-G~Iac!osid~se production induced by TMG added tn the Stationwy pbaS.3 of growth wSS stimulated hv histone Ifta. 2). We can conclude htstone tn our experiments interfered w’tth the process of in‘&tion but it cannot be decided from the present data. what thP Site Bnd mode of tts action we. In our further expertments PTS found that not
ttus
_ that
but
ions also an increasein the can0f ca++ UP to 5 x to-4 M or the P~CSence of Spermb~e (40 ~~g/ml) fully ankgonked the inhibItor” action of histone. We asSun;e that UK effect of htstone is caused not only by its positive CbSrge but alSo by the *is* d its molecule beawe tbe posittvety charged low-molecular wetght agents Itke Mg++, Ca++, and spermine, did not ESSSCss inhibitton of @g&tctosidase Synthesis. The cattonS and Swrrnine SUCCSSS~UUY eonw&S wttb histone for &me electronegSti%‘e btndfk Sites on the Surfaze or within the cells. There 16 sufficient evidence that, e.g., spermti can be hound to a &w&t variety of receptors in the bactertal cell, to phoSpbolipids, cell walls, nucleic acids, etc. (Tabor and Tabor, 1966). A number of bwestfgltors emphasked the lmportlnce of the cell aurlaee, namely of the eytoplaemtc membrane for protein synthe& (see e.g. Toni and Hendlar, 1984). Therefore, tt CMnot be excluded that histone Is bound to the cell surface and interferes v&b the In&&ion w~~ess at Ul level Of transcriDtion or trSnslStson The Surface-bound histone cwld also change the permeabtltty of the cells towards the inducer. Atthcwh this may not be too nrob~ble in ltght of the opposite effect 01 hlsione In the lagarithmx and statimwy phases of growth, the posslhtlity that the histone co&l chDnge the pclrmeabilIty of the cells to Ihe bxiucer is P rest one. HtSto~ could inhtbit Sctive TMG trvlspcwt durine the log &we. It hns been shown In table 1 that the hIStone caa produce cell lysis in UK Stationmy phase. Hence, the ecu w~u Structure mav be drurttwllv &fScted bv hitdone and uermekllity barrI& mSrkedly;educed tn &~-Skiphase. thus ScSountblg for tbS ObServSd stimulatton in #-pale _;~aid~se iretkity. That chwge In the cell wall 6 u\be of primary importpace Is Indicated furt~rr by the fact t&t thy Mg++ Snd CS++ CSn SntagontSa tbe Inbtbttory effecte of h&done. It la well ltnam tint removal of dtwlent cations from the medium by EDTA ssnsitizes the cell Wall of E. cozi to the action Of 1ysowme as well OS makbz It wrmeable to Aetlno&wb~. Cell roll f.wm&btiUy ebimges sbculd be mensured by mevrurlrg the uptake of radbaettve. labelled TMG. An&her Interpretation could be D combltted mech~ntcal yld electrostatic eff& PI famd by YIntar zmd StZstna(leS7) in ~qerbnents in which htetone InhIbited postgermlluttve dSve!opmSnt of bSStuSry SpOreS. The poeeibility thpt histone could penetrate Inte cellrr was confirmed in cell cultures of 801118
only hfg++
cdd0~
the
tionary
higher
organisms
Bukrlnskaya
(Fischer
et IL,
and Wagner,
1964;
1%6).
The hi&me-sttmutated +gatactoatdase inductIon tn the statiorvry phase of 9rowth should be consldared_ The understandiq of the mechanlsm of thir sttmutatlon cwld help m clcutq up home of the cbprges occurring tn thle apeclal ‘duferentiatcd’ state of the cell_
ACKNOWLECGEMENT We would Itke to thank Drs. H. V. Rickenberg, J. Chaloupka, and V. Vinter for voluble commanta and dtecusston. and Miss E. blaanerovl
and ior
Mrs. 0. excellent
Svobcdovl tectmteal
asn1slure.
REFERENCES Tonlno. C. J. 31. ad T-h. It. Rorljn. 1333, rc.nfe of hlnMer) tn yeant. Biocktm. Burger. ht.. E.Pnvllsovl. 1364, Change In lc.xtlon d amylomaltus produced by mutatfcm In Esrhrrickio cdl. Blocham. J. 93. 901.
&i Ike occurBiaykya. Actm
of
Htstone was added to the growing culture after two cell dlvIaIo”s: the tnducer, ‘I’MO(lO-3b%), was added ten minrtee Iater. After a suItable time LntervaI aamoles were taken and treated with cbIoramphe&oI (109 pg/ml) and t&t&e. The aeszy of P-gaLactoaldase In the sunples followed in Ledertmg’a procedure (1950), and wan performed as dee.crUwd prerIoosly (mefI”oVP et al., 1865). Our results are expressed I” te:r”s of epecu*c activity (mp moIes or o-nitrophe”o1 llbelwed from ON% per minute per mg dry weight of the cuIt”re). The percentage of lnhlbltlo” of enzyme i’wmatlon b] histone was cirleulated with regard lo thc speclflc activity of control ceIIn which were “ot treated rith hlr4one. Histone-cbioride ww prepvca from CaIf thymus ad kindly su~lted ky Dr. K. R?Blra Jr. @errnIne tetrahydrc&Iorlde wan purchased from Calblochem. The &r&e were I( glfl of Dr. II. V. Rlcke”berg.
general
3. RESULTS 2. MATERIALS
AND METHODS
The foIIowlng &rains were wed:E.co!l ML 30 l”duclBIe for P-g;lectosldase, andE.cofi ML 308 CO”etlhitive for 8-galaetosldaee, Tho bacteria were inoculated from an overnight culture, and grow” in P reoiprocat‘ng wo&r-bath shaker at 3’ioC in the mineral medium M 56 (Monad et al., 1951) with 5 X 10-3 M II&tow as the sole ~o”rce of carbon. Changes of ooace”tra:lo” of MS+’ and Cat+ WI be indicated In the results.
AND DISCUSSION
It foIlows from the .?~~rime”t preSented I” table 1 that hlsto”e inblbltn the growth of E. coti culture and the i”&clb1e eynthe*ls of #?-galactosldaee. Thla effect depends upon ‘%e cr~neentratie” of Mk ions prewnt in the gro*lh medium. Higher co”ce”tral1oM of I@++ prewlted lhle in. hlbltory actlo” cd hlsto”e, hut the l”h0ltbn of enrymc coiace”!raIo”B of hlstcae used I” our experImco:s (50.409 &I”l) are very hfgh lo comparlsc% with the ctmcentratfcm of 1 &ml whlct wap. deal@&d by IIlrach
famatimM morepronounced thanthe t”htbitt”nof growth.
(1956) aa MuI for E.coli cells. However, HIreoh suepaled the celle In e cltrete-phospbate lmffer end found, in accordance wlth WP reeults, that P higher ccacer.trotlw of poaltlwly charged lone (0.15 - 0.2 M NnCl) blocked the btbal ectlon cd hlstom?. In the concentrrt!nn rage from 5 X 10-4 M to 7.5 x lo-* H Mg+‘, and 1O-4 M CR++ (which te the ueuel cmceatratton of Ca++ ln the mineral
medium M 55) histonc at a concentration of 400 pg/ml prcfereritialty lnhiblted P-galactasidase syntheale without pronounced inhibition of growth. On the basis of this observation we attempted to matntatn in the f~UowIng expeFimente an optimal ratlo of histone: Mg++, which would exert the most pronounced effect on the activity of Inducible ,%galacto,sidase Without influencing the growth of the cultwe. One of these experlmanta IS presented in fig. 1. It can be seen that hletone lnhlbited the Inducible synthesis of P-galactostdase during the logarithmic phase of growth. In the stationary phase, cells treated sltb hlstone reached the fame speclflc activity of enzyme as the control and, mo~over, a stimulatlon of enzyme syrrthesls over the control level was observed In some earlier communications Srow our laboratory (Burger and Pavlas0va, 1954: NeEinov% et al., 1965; NeClnnv6 and Burger, 1965: Lefk&t8 and Burger, 1805) we reported that both in uflro or in uiuo, histone cculd liberate /J-&Plectosidase and amylomalhse bound to cell etructu.es in come E. coli mutants. Hence we cmld the posslbllity that the histone Mlbltion of s-g.llnctostdaseformatlon wee not due to an achtal Inhibitton of its eydhesis. We postu:ated the alternattve explana” m that histone in
notexclude
the logarithmic phsse caused S btndtng of the enzyme to Cell structures and tts cansequent bratttvatlon. 0” the other hand, In the StStionary phase, P-gaiaofostdase ~1s liberated from these structures by the htstone present. This pcssibiltty was rules out by the following SVidellCe: a) In thS presence of cblorSmphenlco1 added to the induced culture at the beginning of the stattonary ,,hnse of growth, no further enzyme pro&ction to.& place or, bl‘2tiler wordS, “Cl eneyme was ‘liberated’ by histone in the stationary phase.
b) The ~ratucttcn af constitutive @-gglactoSid1se In the ML 308 BtraIn wSS not irdluenced by bistone either tn the logSrtthmte or in the Stationary phase. c) p-G~Iac!osid~se production induced by TMG added tn the Stationwy pbaS.3 of growth wSS stimulated hv histone Ifta. 2). We can conclude htstone tn our experiments interfered w’tth the process of in‘&tion but it cannot be decided from the present data. what thP Site Bnd mode of tts action we. In our further expertments PTS found that not
ttus
_ that
but
ions also an increasein the can0f ca++ UP to 5 x to-4 M or the P~CSence of Spermb~e (40 ~~g/ml) fully ankgonked the inhibItor” action of histone. We asSun;e that UK effect of htstone is caused not only by its positive CbSrge but alSo by the *is* d its molecule beawe tbe posittvety charged low-molecular wetght agents Itke Mg++, Ca++, and spermine, did not ESSSCss inhibitton of @g&tctosidase Synthesis. The cattonS and Swrrnine SUCCSSS~UUY eonw&S wttb histone for &me electronegSti%‘e btndfk Sites on the Surfaze or within the cells. There 16 sufficient evidence that, e.g., spermti can be hound to a &w&t variety of receptors in the bactertal cell, to phoSpbolipids, cell walls, nucleic acids, etc. (Tabor and Tabor, 1966). A number of bwestfgltors emphasked the lmportlnce of the cell aurlaee, namely of the eytoplaemtc membrane for protein synthe& (see e.g. Toni and Hendlar, 1984). Therefore, tt CMnot be excluded that histone Is bound to the cell surface and interferes v&b the In&&ion w~~ess at Ul level Of transcriDtion or trSnslStson The Surface-bound histone cwld also change the permeabtltty of the cells towards the inducer. Atthcwh this may not be too nrob~ble in ltght of the opposite effect 01 hlsione In the lagarithmx and statimwy phases of growth, the posslhtlity that the histone co&l chDnge the pclrmeabilIty of the cells to Ihe bxiucer is P rest one. HtSto~ could inhtbit Sctive TMG trvlspcwt durine the log &we. It hns been shown In table 1 that the hIStone caa produce cell lysis in UK Stationmy phase. Hence, the ecu w~u Structure mav be drurttwllv &fScted bv hitdone and uermekllity barrI& mSrkedly;educed tn &~-Skiphase. thus ScSountblg for tbS ObServSd stimulatton in #-pale _;~aid~se iretkity. That chwge In the cell wall 6 u\be of primary importpace Is Indicated furt~rr by the fact t&t thy Mg++ Snd CS++ CSn SntagontSa tbe Inbtbttory effecte of h&done. It la well ltnam tint removal of dtwlent cations from the medium by EDTA ssnsitizes the cell Wall of E. cozi to the action Of 1ysowme as well OS makbz It wrmeable to Aetlno&wb~. Cell roll f.wm&btiUy ebimges sbculd be mensured by mevrurlrg the uptake of radbaettve. labelled TMG. An&her Interpretation could be D combltted mech~ntcal yld electrostatic eff& PI famd by YIntar zmd StZstna(leS7) in ~qerbnents in which htetone InhIbited postgermlluttve dSve!opmSnt of bSStuSry SpOreS. The poeeibility thpt histone could penetrate Inte cellrr was confirmed in cell cultures of 801118
only hfg++
cdd0~
the
tionary
higher
organisms
Bukrlnskaya
(Fischer
et IL,
and Wagner,
1964;
1%6).
The hi&me-sttmutated +gatactoatdase inductIon tn the statiorvry phase of 9rowth should be consldared_ The understandiq of the mechanlsm of thir sttmutatlon cwld help m clcutq up home of the cbprges occurring tn thle apeclal ‘duferentiatcd’ state of the cell_
ACKNOWLECGEMENT We would Itke to thank Drs. H. V. Rickenberg, J. Chaloupka, and V. Vinter for voluble commanta and dtecusston. and Miss E. blaanerovl
and ior
Mrs. 0. excellent
Svobcdovl tectmteal
asn1slure.
REFERENCES Tonlno. C. J. 31. ad T-h. It. Rorljn. 1333, rc.nfe of hlnMer) tn yeant. Biocktm. Burger. ht.. E.Pnvllsovl. 1364, Change In lc.xtlon d amylomaltus produced by mutatfcm In Esrhrrickio cdl. Blocham. J. 93. 901.
&i Ike occurBiaykya. Actm