Relationship between competence and doubling time in bacillus subtilis cultures

Relationship between competence and doubling time in bacillus subtilis cultures

$~~n~ieat~ii ~II' ~' 1141-1150, 1970 . Pergamon Preen HIIJlIOHBHIP HS111HiH COIIpHli11CH ~ DOOHLIIfG lI1R Ilf 8DHl1LI4 COLlHMB 3dCZI ~ H. Lapes...

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~II' ~'

1141-1150, 1970 .

Pergamon Preen



3dCZI ~

H. Lapes, t. Perblis and Ma.l. Pires-~ireHa Isstitsb MJaise perrine de MiorobioloHia del C.S.I.C., Jeagsia Conta 39, Madrid - 8, épais

(Received 20 July 1970 ; in ünal form 3 August 1970) 11sor aibatier iu been paid b the eeespetgoeo state tellovirs tie stsdies is paetio traastorsatioa by drert et al . (Z~ . Hoto6ltiss (6) ban detisititelp saowd it s~aorroaons ~rovtr of Diuloosaoss fiat a pre of oospebnoe appears daris8 all aserth . Ia pwsroooaai (18) sad sore reoeatlp is strepbooooi (1H, 13) it bare shova the role o! a speoitio oospetgt factor ( aotivabrr) . is B, sabtilis, fair cospebsoe stab si~rt b. arsooiated viii the spornLtios proowss altroasi this it s ssbjeot of eoatrorerrp (181 . Certaialt 1lesbr (11~ wueated fiat the oospobaoe ps direotl? related b a restis~ oeil stab. Hott sad 1lilsoa (3) st:d~iss tie iatlneaoe of setabolio sad antritiorsl taotors oa ire developpnt et oospebaoe is setiral strains of H. sabtilis oosol:ded tact the oospebaoe pve, is batch ealtnres, is not sheeted b~ the nab e! srovti dnrias the ezpoaeatial phase vhereaa Horvat6 (d~ has observed a relatiorsrip betwea seaeratioa tire sad oospetoaw depeadir~ w several !actors . Hoeanso of these eoatroversial tirdiap w have studied the oospeteace state of H= sabtilis is the lob phase sad its possible voriatioas as a siasle mastics of the dosblir~ tire. 1~aterial sad Methods Eaebrial straiw . H. sabtilis strain 168 (wilt type) vas aced, as the donor strain. !he reoipsgt baobria ras H. sabtilis SH-2â 1141



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(Lisa tr~~) (10) a doable aazotreph lraa strain 168 !or the tvo listed asters . Beth bacterial strains w» kept en nutrient a8ar shah . O:arti siedia . 8ott and Yilsos's nediaa (3) vas roatiaelT aced !er dereloptsent o! cupeteaae but is sane esperirenb either the sistidine er trpptophaa coacealratiou wre ohaased. Ifatrieat sßar plats wre ased to estisate the total aasMr e! ersaaissis sad both transtersed sad doable retrosw~taats erßaaiws wre aonated on the sinisal siediu e! daapeatopetilos and Spisisea (1) sliß4tl? soditied . 8iaßie retrewtaats (hiss trl~ er hiss tr~ 3) wre controlled oa the sale siaisal ~ediu ooiatainina either histidine or trtptophaa as regaired . Orovth is coatinnoas onlt~re . ~ coatianow anlt~re slsta similar to the Boseaber6er and Bisdea's sedel vas ased (14) . Borlciaß rolose in the ahe~wtat vu 300 ~1 and, in addition to the lateral outlet tie spparatns vas equipped with another wple deriae in order to pt fresh anltare vithoat delay !or transtor~ias purposes . fir vas supplied at a rate o! approziratel~ 1 1/hr sad te~peratnre vas 37o C = O.S . ?he steady-states wre eoatrolled b! O .D . seasarepat at 610 s~. viii a Bausch 8 Lonb Speatroaia a0 sad pß variations in the sedia wre estimated with a Colesaa mod. lletrioa II . In order to taor the level o! the lisitias laator !a the different rags, sesigaaatitative siorobioloßioal detersinatioas o! the histidiae wre carried eat (1?) . ~lignots o! the anltare wre filter-sterilised bT usia~ s O.Z2~t. asbraae Millipore filter sad added to the sinisal ~ediu vithoat histidiae sad the bacterial ~rovti vas lolloved aephele~etricall~. Psenaration o! traaslor~iac Dli~ . Bacterial DNA vas obtained, at the end o! the ezpoaential phue lro~ B. sabtilis strain 168 srova is Penawa? sediu fellovias the sethod o! Marsar (D) and farther phenol treatpat vas need to avoid spore coatuiaatioa Lß) . Coaoeatratioas

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of DNt wre censured at 980 r~ is a 8"ahran D,U . Spectrophotoreter . Protein content of D/t preparations ras alrys less than ~ as dete :Tined br the athod of Lorry et al (8) . Grorth o! recioent cnltnres . Corpetenoe of recipient cnltnres ws studied in either batch or coatiaaons cnltnres . To stadT corpetence is baton cnltnres bacteria wre Hrora in 100 rl nepheloswtrio clashs aocordina to the rethod of Bott cad 1lilson (3)i histidine or tr~ptophar concentrations ranain~ troc 100 to 1 .â6 r6/1 wre need and trasstorrations rare carried ont at ditterent worth phases . To stade corpetence is a continuons caltnre the receptor strain irw a oyernisht ~rorn culture in PenassaT rediw vas csntritapd cashed trios with riairal salt solution and inoculated to the cherostat to ~i~e an initial O,D . of 0 .07 at 610 r~ . titer the culture Brow to its causal density is the cherostat, the continuons culture ezperirent vas started rith suerai dilution rates . Transtorratioa orooedure . The traastorration riztures ooataiaia~ 0 .9 rl of recipient corpetent bacteria + 0,1 rl of DNt (7 to 10


in test tsbes (30 : 900 a) wre incubated at 379 C to 280 rpr in a i~er Hrussriak shaker yodel G-25 . tuer 20 sin of inonbation the reaction ras stopped b~ addition of 100 ~tl.~ (0,1 rl) of Dhase (YOrthia~ton) and shakias ras continued for 10 rial the appropiated dilations sere prepared is saline (pit 7 .0) and plated os sinisai udia . In another set of e:perirents 0 .1 rl of DNt rare treated rich DHase is the presence of ô

,roles of ll~ Cl at 37Q C for


sin before

the addition to the caspeteat cells, incubated at 37Q C for 30 sir and inoculated on sinisai nedinr . Traastorratioa tregneao~ vu obtained w a rate betreen nwier o! doable transtorrants (hisâ tr~Z) per rl tires 100 retered to the total umber of cells per rl .



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Daria~ the inoabation of tie trautoraatioa aiztaren parallel tenu with the oonpetent oohs, wse carried est on uphelee~otrie tladrs and the O,D. w ~easnred to teat the puration tiu variations ud to iaev rhea tie udia bepa to be oàaasted . Desulb aad Diseasiioa i!e vasiatiou la the ao~ber et traastor~ant ter D. stbtilis srevia~ L batches wrier wversl ooaditioss wre stadied is preliaisar~ ezpesineatr. tip 1 and Z illsstrate the transtorubilitt o! ~. sobtilis at as earlt statienar~ phew tree oaltnres rich different histidine aad tr~ptephaa eoneentratioas inoo~lated with as iaitial O,D, o! 0.07 at d10 n/u, (abeat 107 eoloq lorrrs/al) .

w F <

pip . 1 and Z ltelatienship betwea e:ponentiai p~orth rates ( ~------fl ) aad tsustesaatioa tregwno~ ( H ) eonditioaed ~ dittesent sobstrate oonoontratio®s o! tvo li~itia~ faeters ia 1. pbtilis

Yol. 9, No. 20

COMPETENCE IId B. subtilis


lsaaatorwtiea le~eL depead w boti cuceatratioa ad aatare K rabstratea aai tie~ wre rhora to w ~ere 1i~itaat !er birtidiae st t~ raspa rred. It L aot penible te der lrea thw ewalta it eities liaitiai taster oeawatratios or e:pewrtial srertb rater (/IL a) are reapowble ter these trantorrias sariatioar . Ot~r esperiaeata wn portorrod ariss iaooala aa abore (lip. S asi 4) t" laer the ~asiatioaa o! tbe ooapeteaoe raq q raias ae~eral biatidiw coaaeatratiera t. pt dittereat ~a.


ris. s

ia O.n, ot ~D ~!¢°~ at ditt~reat oeaoe~tratiosr o! hiatidire lir/~i/u1 : canea ai . 100, as . 60, C~~ b o " 1Z.6, d . d .sô, e " " 1.a6 . Circled ar~6era are tie aaaplirs poiata to detesriae tbe w~e et oorpeteaoe L traartoraatiea . re ferai, v portalatei (3) that the aa:iasa de elepaeat et eoyeteaoe appears 3 br after tho ~ad of the ezpoaeatial srerth pbw ries venins st certain coaowtratieaa et the liaitias factor (biatidia)

(pis. 4) while at lover leselr the ooap"toaee ra"N beeaae aarrowr: the peal ei eoapeteaoe appears betoro and oo~peteaoe laiatei est gsietos. V w poiatei eat before it wa dittioalt to b'aer it the ~ariatiear it trantorrias eapacitp coald be attribated eitber'te the ditteroat

srertb rato (~u.) er to the lever coaoentratio~ar of the liaitias taster which cirer rire to a asdic rwpidlp e~aorted or to bette sisoaartaasea . Oa the otbor band, them ooaditioaa a. veil as thow nei b~ Horrath (4) casts sere downs about the iatlaeroe of factors chers



Vol. 9, No .20

tbaw /Lt ~ ; !or ernple oatabolie sabstances soosnwlated iw the rdia dependiss e" enltwral eonditiou (û) .

ris . ~ ~ariations in the eo~peteaae vase o! D . sabtilis 83-Zâ (bis1 trr Q l aeoerdias to tbe nazlnal srorti rate salau aad üstiäine ooaoentrations in the ceaditioas sises is lisnre 3 . Ia order to amid the intlwenee o! sweb catabolic prodnets on ~, sabtilia translesaatioa u wll u to pt different doablins tir coast~at !er less periods of tir w periorw esperirnts ritb the chewostat . Obsiowsl~, all the trawstorwations wre carried oat vit6 the pepwlatios is ezpoaential phase o! srorti is vhicb the conpeteaee ru net at the naziwon lenl . Ogee a stead! slate ras established the O .D . remised practioail! eeastant dnrins the ilor rate (f is . a i . In each rnn w controlled the lose! of the linitins !actor rewained b! the aicrobiolosical procedare described . eistidir ras teser ezhansted in aa~ o! the three conoeatsatioss need . ~ parallel twsbidisetric test ras carried cwt in nephelortsia llasi rhüe the twnt~ master traasiorwatioa procedure ru

Vol. 9, No. 20



deco. this vas pertorpd is order to iaov thdt~t.roaaird useable is the oaerpeteat call suple tales trot the oheaostat for at lent ep hear. The rags for aq~ Binn steadT state case woes shorter that 48 hr and eoatinnons operatiow with the eheswstat hs~e bees carried oa darias 10 - 17 dais; PB ulnas lspt Mtwea 6.8 to 7.0 .

piB. 8 Stead~-state srovth of B . anbtili= 8B-~ (hin- trp~) viii three histidiae waaatration u .liaitiaB tae~er ( ~ 8; ~ 18i / 80 ~i/1) . Dilntioa ratet d . 0,183, B . O .lY, C . 0 .173, D . 0,40 aad : . 0 .833 6s-1 The rate of siasle retraantaat (hiss trp~ er hL~ trp~) ran`ed betwea 0.4 sad

1 .8 z 10~


0 .8 z 10-11 .

rhile doable retrawtaats (hiss t~1) cars

The rariatioaa of traostoraia8 capacity of

BB .

snbtili~ (hiss tr>-1 )

is inaction of doabliaa tine are presented is rid. Q shevia6 olearl~ s olose relationship betwea de~sloppat of oaspeteaw sad the ~rwti

rate with a peat at a donblin6 tip of abort 1.8 hs1. The sap segpaw of facts care toaaded when asia~ ditterent a~onnts of the li~itaat aatrieat althoasht the levels of transtos~atiea tregaewies, vithia the sane dotbliad tine, thanes proporciooalip awordind to the histidiw wawatratiou .

114 8



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~elatiensüp Mtwen doubling tine and transformation ireogmenq in H . subtilis SD-26 (hiss tr>Z) groring is chemostat rith tires different hislidine aonoentrations (a0, 2ô and 6 mg/1) as limiting factor . $ach point represents the averap value of three determinations . ~t different llor rates, some differences in the bacterial sips wre observed sicroseopicallt by the Gram stain . It is pnerall~ thomght that a hight level of competence arise rhea the bacterial grorth rate is affected bt ohaagiag the cells irem a eomple: media to a deüsed one (13, 7) ; is agreement Tith that vas toned (16) defective traasformatimg eapacit~ is matants rich an altered grorth rate . In addition, other Torhers (16,20) sugpsted that cell-Tall and membrane changes are related to the competence state . X11 these !lading are 1n accordance Tith the results w have shorn since this competence rave of the D . subtilis groring in a continuous aalture, as a fanetion of

might M related to the changes observed is the bacte-

rial length at different llor rates . prelisiaart test seen to saggest that these morphological variaLions might affect to the surface/volume ratio and consegaentl~ to the structural disposition of the outer layers of the recipent cell . This might alter its ptysieo-chemical properties and thus its permeability

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to iniearate the doaer Dam . Finally, osr ezperi~ental aonditioss let o to aeatirs the Hers of Hornth (4) aMnt the relationship Mtwen pwration tiu and tie de~elop~ent of ao~petenoe rithoat the intlneaae of tasters other than p~orth rate. Baste the intlnenae of the donblias tiae on the cospetenw state of a double-an :otroph sntant of H. sabtilis has Men stadied . On batch oaltnres it ras not possible to establish each a relationship Moause of the intlnence of amore than one factor . De~elopaent of aarpetenoe in the losaritaia phase of worth ras also pestor~ed in a coatinnons aultnse s~stes. ~n optimal transtor~atioa aapacit~ ras reached at a donblin~ tine of abort 2.a hi1 b~ nsia~ different tlor~rates. ~ov1~dt~sen~s Ye rish to erpress onr aratitnde to Dr. D, Yasgnes !or his interest in the preparation oi this sannsaript. The technieal sasistanoe ot hiss Na, ?. ~lda and Ms, L, dsl Poso is 6ratetall~ aoinorled~ed . Iteterenas 1 . C . dädGN08?OPOIIIAB and J . SPIZIZ81f, J. Haateriol . ¢j,~ 741 (19d1) 2. 0 .?. dY6HT, C.M. McLHOD and M, McC1itTT, J . Lzn . Med. ~, 2a (1444) 3. 1C,F, HOTT and G.A. YILSON, J . Bacteriol . ~4, a62 (1967) 4. 8. HORY~?H, J . ten . Hicrobiol . ~8, 21a (1987) a. H,D . HOTCHYI88, Proc. Natl . Decd. Sa. D.B. ~, 49 (19a4) 8. Y .B, âIRHT, Hioches. J. 6~, 49a (19a7) 7 . C,G, LFANUtD, D.=. 111TTHEI$ ! 11.J. 1141THEI8 and R.D, HOIIBHrHIGH?, J. Haateriol . 88, 220 (19d4) 8 . O,H. LOrB ?, N .J. It08H880UGH, 1.L. F~ and 1t.J. ~IIDdLL, d C~is . 103, 28a (19a1) 9. d. 11~HlNlt, J. Mol . Hiol . 3, 208 (1981)

b o



lo . s .Y . rrsrsQ, M . swA~sr ana J . LeDSSasra,

Vol . 9, No. 20 ~, a29 (1963)

11 . D .Y, rr8lsQ, J . Daaterioi . ~, 867 (1964) 12 . 1t . PuU1J, J . saoteriel . ~0, 1â01 (196a) 13 . D . PADULA, P . WT and L .lt . BPirCiR, Cad . J . Miorobiei . j4, 34a (iD70) 14 . R .P . DQB=1®sß(iDß and B .A . sLBDSr, J . sen . 111arebiol . ~, 726 (1960) la . r .l . 8P=rCSit add 1t,11 . ~itSIO!!, J . Baoteriel . ~0, 911 (1966) 16 . J . BPIZIZrr, D .s . 1tSILLr and A .r, sYAlIB, Aea . De~ . Miarebiol . ~0 , 371 (18d6) 17 . D .P . 8lsELt, r .s. BAQBY3lLICr, M .B . ftDrroLDB add C .A . B1011UIr, J . aiol . Càu . ~, â33 (1~9) 18 . A . lO11A$Z add It .D . HOlC~IBB, Proa . rat . Aaad . 8ai . Q .B . ~, 480 (1964) 19 . l .s . roU11G aad J . SPIZIZEr, J . Saateriei . ~,, 823 (1961) 90 . r .s . routa, rats. ~, io4 (1Y6ô)