DNA-DNA Homology Between Lactic Streptococci and Their Temperate and Lytic Phages

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1 APPLED AND ENVRONMENTAL MCROBOLOGY, My 1984, p /84/5131-8$2./ Copyright C) 1984, Americn Society for Microiology Vol. 47, No. 5 DNA-DNA Homology Between Lctic Streptococci nd Their Temperte nd Lytic Phges AUDREY W. JARVS New Zelnd Diry Reserch nstitute, Plmerston North, New Zelnd Received 31 Octoer 1983/Accepted 13 Ferury 1984 Temperte phges were induced from Streptococcus cremoris Rl, BK5, nd 134. DNA from the three induced phges ws shown to e homologous with prophge DNA in the cteril chromosomes of their lysogenic hosts y the Southern lot hyridiztion technique. 32P-leled DNA from 11 lytic phges which hd een isolted on cheese strters ws similrly hyridized with DNA from 36 strins of lctic streptococci. No significnt homology ws detected etween the phge nd cteril DNA. Phges nd lctic streptococci used included phges isolted in recently opened cheese plnt nd ll the strter strins used in the plnt since it commenced opertion. The three temperte phges were compred y DNA- DNA hyridiztions with 25 lytic phges isolted on cheese strters. Little or no homology ws found etween DNA from the temperte nd lytic phges. n contrst, temperte phges showed prtil reltionship with one nother. Temperte phge DNA lso showed prtil homology with DNA from numer of strins of lctic streptococci, mny of which hve een shown to e lysogenic. This suggests tht mny temperte phges in lctic streptococci my e relted to one nother nd therefore my e homoimmune with one nother. These findings indicte tht the relese of temperte phges from strter cells currently in use is unlikely to e the predominnt source of lytic phges in cheese plnts. t hs een shown y numerous workers tht lctic streptococcl strins used s strters in cheese mnufcture re lysogenic in tht induction of these strins leds to the relese of phges which cn e seen y electron microscopy (2, 3, 7, 12, 13, 17). n few instnces, notly for Streptococcus cremoris R, (12), S. cremoris BK5, 187, 188, nd 189, nd Streptococcus lctis C2 (3), the induced phges hve een propgted on strins of lctic streptococci. Therefore, lysogenic strter strins must e regrded s potentil source of phges in cheese plnts (2, 3, 7, 13, 17). However, despite the mny ttempts to propgte induced lctic streptococcl phges, indictor strins hve not een found for the mjority of the phges seen y electron microscopy. This leves open the question whether some of these phges seen fter induction re defective or whether the strins tested s indictor strins re not cple of supporting the growth of these prticlr phges. The phges seen fter induction of strter strins re most frequently of the smll isometric type (5), similr in morphology to the phge species most commonly isolted from cheese plnts. f phges relesed from lysogenic strters re n importnt source of the phges detected in cheese plnts, it is hrd to understnd why induced phges hve proved to e so difficult to propgte on strter strins. n previous study (6), DNA from lytic lctic streptococcl phges ws chrcterized, nd the phges were differentited from one nother y DNA-DNA homology. The present study ws undertken to provide definitive dt on the reltionships etween lytic phges isolted from cheese plnts nd temperte phges present in strter cells. First, DNA from three induced phges ws compred y DNA- DNA hyridiztion with DNA from 25 phges isolted from cheese wheys. Second, it ws shown tht prophge DNA could e detected in the DNA of lysogenic cteril cells y Southern lot hyridiztion. This technique ws then used to determine whether DNA from 11 lytic phges ws present s prophge DNA in 36 strins of lctic streptococci. 131 MATERALS AND METHODS Bcteril strins. All of the strins studied re in the New Zelnd Diry Reserch nstitute collection nd re listed in Tle 1. Phges. Three phges were induced y mitomycin C (MC) induction (12) nd re nmed from their lysogenic hosts followed y the letter T (thus, 134-T from S. cremoris 134). The remining phges were isolted from cheese wheys nd re designted y their numers in the New Zelnd Diry Reserch nstitute collection followed y the numer of their propgting cteril strins in prenthesis. Preprtion of phge DNA. The preprtion of phge DNA nd leling of DNA with [32P]CTP or [32P]ATP were crried out s descried erlier (6). Preprtion of cteril DNA. Cultures (4 liters) were grown for 2 h t 22 C in M17 roth (16) nd hrvested y centrifugtion. Lysis ws crried out s descried y Klenhmmer et l. (1). At the completion of lysis, sodium perchlorte ws dded to finl concentrtion of 1 M, nd the mixture ws extrcted with n equl volume of chloroform-isomyl lcohol. The queous lyer contining the DNA ws removed, nd the DNA ws precipitted with 2 volumes of cold ethnol. DNA ws dissolved in 1 ml of dilute sodium sline citrte. RNse (Sigm Chemicl Co., St. Louis, Mo.) ws dded to finl concentrtion of 5 jig/ml, nd fter 3 min of incution t 37 C, protese (Sigm) ws dded to finl concentrtion of 2 jig/ml. After further incution of 1 h t 37 C, the solution ws extrcted twice with Tris-sturted redistilled phenol (BDH Chemicl Co.) (5 mm Tris [ph 8]) nd twice with chloroform-isomyl lchohol. The queous phse contining the DNA ws dilyzed overnight ginst 4 liters of uffer (2 mm Tris, 2 mm NCl, 1 mm EDTA [ph 8]) nd stored t -7 C. Approximtely 6 ml of DNA solution contining etween 6 nd 2,,ig of DNA per ml ws otined y this method. Purity ws ssessed y sorption spectr t wve-

2 132 JARVS Phge TABLE 1. Lytic phges Propgting strin Smll isometric phges type or AM ,c C Smll isometric phges type c E Prolte phges type d ML ,c c ,c ~~~~~~~~~566 Lrge isometric phges type e ,c C ,c "All propgting strins re S. cremoris except ML3, which is S. lctis. DNAs from these phges were leled with 32P nd used s proes with R-T nd 134-T DNA. 'DNAs from these phges were hyridized with 32P-leled DNA from BK5-T lengths of etween 21 nd 3 nm (9), nd rtios of redings t 258/23 nd 258/28 nm of greter thn 1.8 were regrded s stisfctory. Chemicls. Deoxycytidine 5'-[-32P]triphosphte nd deoxydenosine 5'-[-32P]triphosphte (specific ctivity, 2, to 3, Ci/mmol) were otined from Amershm Austrli Pty. Ltd., Aucklnd, New Zelnd. DNA polymerse (lrge frgment, Klenow enzyme) ws otined from Boehringer Mnnheim, GmH, West Germny. Restriction enzyme EcoR ws kindly supplied y the Virology Unit, Mssey University, Plmerston North New Zelnd, nd Hindll ws otined from Bethesd Reserch Lortories, nc., Rockville, Md. Gel electrophoresis for hyridiztion etween phge DNAs. Restriction endonuclese digests of phge DNA (.6,ug) were frctionted in horizontl sl gel pprtus in.8% grose (Sigm) in Tris-cette uffer (ph 7.8) s descried erlier (6). Gels were stined with 4,ug of ethidium romide per ml for 1 min, visulized under UV light, nd photogrphed. Gel electrophoresis for hyridiztion of phge DNA to cteril DNA. EcoR digests of cteril DNA (1.25,ug) nd phge DNA (.125,ug) were frctionted on.8% grose gels s descried ove. This rtio of DNAs ws chosen ecuse the moleculr weight (MW) of lctic streptococcl APPL. ENVRON. MCROBOL. DNA is pproximtely 2,7 kilose pirs (8), nd the MW of lctic streptococcl phge DNA is pproximtely 3 kilose pirs (6). Therefore, if one cteril genome contins one prophge, pproximtely 1 times the mount of cteril DNA compred to phge DNA is required to give equivlent quntities of phge DNA. DNA-DNA hyridiztion. The Southern lot technique (15) ws used to trnsfer DNA frgments to nitrocellulose filters of.45-,um porosity (BA-85; Schleicher & Schull, GmH, Dssel, West Germny). 32P-leled DNA (2 x 15 to 5 x 15 cpm per filter) ws then hyridized with the DNA on the filter s descried erlier (6). After hyridiztion hd een llowed to proceed t 6 C for 2 h, the filters were wshed nd used to expose X-ry film cked y Cronex (Du Pont Co., Wilmington, Del.) Lightning-Plus intensifying screen. Acceptle results were otined fter 1 to 5 dys of exposure t -6 C. Severl hyridiztions were lso crried out t 55 C for 2 h. Results were similr to those otined t 6C. Plsmid preprtions. Plsmids were extrcted y the method of Lelnc nd Lee (11) from 35-ml 5-h culture of cells in M17 roth. The plsmids were seprted on.8% grose gels s descried for phge DNA frctiontion. Between 1 nd 1 plsmids were detected in the strins exmined. Electron microscopy. Electron microscopy of phges ws crried out s descried erlier (5). RESULTS Phges induced from lysogenic strins. Lystes produced y MC induction of S. cremoris 134, Rl, nd BK5 contined phges which could redily e seen under n electron microscope (Fig. 1). Phges 134-T nd R1-T were of the smll isometric type (5), hving isometric heds, (dimeter, 56 nd 53 nm) nd til lengths of 13 nd 126 nm, respectively. Phge 134-T hd se plte, which could not e seen in R1-T. Phge BK5-T hd similr hed (dimeter, 54 nm) nd til 232 nm long. Phges 134-T nd R1-T were concentrted directly from MC lystes, nd the phges were purified nd DNA ws extrcted s descried ove. t ws not possile to otin sufficient BK5-T from the MC lyste for DNA preprtion, nd BK5-T ws grown on S. cremoris H2 efore concentrtion, purifiction, nd DNA extrction. DNA-DNA hyridiztion mong temperte phges. EcoR digests of DNA from ech of the three temperte phges were seprted y gel electrophoresis (Fig. 2). Hyridiztion of 32P-leled DNA from phge R1-T (Fig. 2, lne 1) showed prtil homology with DNA from phge 134-T (lne 2) nd from phge BK5-T (lne 3). Similrly, prtil homology ws seen etween the temperte phge DNAs when DNA from 134-T or BK5-T ws used s the proe (Tle 2). The numer of nds, given in Tle 2, of DNA leled y homologous DNA indictes the numer of nds present on the gel for ech DNA. Thus, gel electrophoresis of the restriction endonuclese digest of DNA from phge R1-T produced seven mjor nds nd two minor nds of DNA, of which three showed prtil homology with 134-T DNA. DNA-DNA hyridiztion etween temperte phges nd phges isolted from cheese plnts. 32P-leled DNAs from temperte phges 134-T nd R1-T were hyridized to EcoR digests of DNA (Fig. 3) from 25 lytic phges (Tle 1) which hd een isolted on strter strins. These 25 phges represented the four phge species descried erlier (6) for lctic streptococcl phges, nmely, phges with isometric cpsids, termed smll isometric phges types or (12 phges) nd smll isometric phges type c (two phges), phges with

3 VOL. 47, 1984 LACTC STREPTOCOCC AND THER PHAGES 133 loo nm FG. 1. Electron microgrphs of temperte phges. () phge 134-T; () phge BK5-T; (c) phge R-T. prolte cpsids, termed prolte phges type d (seven phges), nd phges with isometric cpsids, termed lrge isometric phges type e (four phges). DNA from phge Rl- T (Fig. 3, lne 5) showed prtil homology with DNA from three phges of similr morphology (Fig. 3). These phges A were 853 (AM1) (lne 8, one of four DNA nds leled), 19 (168) (lne 6, one of six DNA nds leled), nd 1367 (14) (lne 1, one nd of two leled). No homology ws found etween DNA from R1-T nd the remining phges. Phges R1-T nd 853 (AM1) cn propgte on S. cremoris AM1, nd it is possile tht they possess common DNA sequence which llows them to grow in the sme host. They re unrelted serologiclly y neutrliztion tests (unpulished dt), so tht it is unlikely tht they shre common til ntigen. 32P-leled DNA from phge 134-T showed homology with one high-mw frgment in the DNAs of the two lrge isometric phges 1283 (134) nd 1299 (134). The homology ws not due to contminting cteril DNA from S. cremoris 134 in the phge DNA preprtion, since neither 1283 (134) nor 1299 (134) showed ny homology with S. cremoris 134 DNA. No homology ws detected etween DNA from 134-T nd the remining phges. 32P-leled DNA from BK5-T ws hyridized with DNA from 1 lytic phges (Tle 1). No nds of homology were detected. Hyridiztion etween temperte nd lytic phges ws lso crried out using lytic phge DNAs s proes. Leled DNAs from 14 phges (Tle 1) representing the four phge species were hyridized to DNAs from the three temperte FG. 2. () Gel electrophoresis of EcoR digests of temperte phge DNAs. () Autordiogrm prepred fter hyridiztion of 32p_ leled DNA from phge R1-T to DNA frgments. Lne 1, R1-T; lne 2, 134-T; lne 3, BK5-T. Bnds of homology were seen t A nd B (lne 3) nd C, D, nd E (lne 2). TABLE 2. Homology detected mong DNA frgments from EcoR digests of temperte phge DNAs Source of No. of nds for the following sources of unleled ~~~~~~2P-leled DNA': DNA R,-T 134-T BK5-T R1-T 7 (2) 2 2 (2) 134-T BK5-T (1) Numers in prentheses indicte wekly leled nds.

4 134 JARVS APPL. ENVRON. MCROBOL _- ~w il FG. 3. () Gel electrophoresis of EcoR digests of temperte phge R1-T nd lytic smll isometric phges. () Autordiogrm prepred fter hyridiztion of 32P-leled DNA from R1-T with DNA frgments. Lne 1, 1367 (14); lne 2, 124 (266); lne 3, 1358 (582); lne 4, 936 (158); lne 5, R1-T; lne 6, 19 (168); lne 7, 929 (266); lne 8, 853 (AM1). phges. 32P-leled DNA from phge 1299 (134) showed homology with one high MW nd in 134-T DNA. 32pleled DNA from phge 124 (266)'showed wek homology with one nd in BK5-T DNA. Otherwise there ws no homology etween DNA from the lytic nd temperte phges. Effect of propgtion of temperte phge R,-T on DNA homology. Phges 134-T nd R1-T were concentrted nd purified directly from MC-induced lystes for DNA preprtion. No indictor strins hve een found for 134-T. However, phge R1-T will grow on severl indictor strins. To determine whether propgtion of the induced phge through indictor strins ltered the hyridiztion of the phge DNA, R1-T ws grown on S. cremoris AM1 nd 368. The phges were then purified, nd DNA ws extrcted s efore. EcoR digests of DNA from phges R1-T (MC) (phge concentrted directly from MC lyste), R1-T (AM1), R1-T (368), nd 853 (AM,) were frctionted on.8% grose gel (Fig. 4). DNA from phge 853 (AM1) ws included ecuse it ws similr in morphology to tht of phge R1-T, oth R1-T nd 853 (AM1) grow on S. cremoris AM1, nd prtil homology ws found etween these phges. 32P-leled DNA from phge R1-T ws hyridized to EcoR-digested DNA from phges R-T (MC), R1-T (AM1), nd R1-T (368). All the DNA nds of R1-T (MC), R1-T (AM1), nd R1-T (368) were leled y the proe (Fig. 4), wheres only one nd of 853 (AM,) DNA ws prtilly leled. Gel electrophoresis ptterns of EcoR digests of DNAs from R1-T (MC), R1-T (AM1), nd R1-T (368) (Fig. 4) were identicl to one nother nd differed mrkedly from tht of 853 (AM1). Titers of phge R1-T (AM1) nd R1-T (368) were similr on S. cremoris AM1 nd 368; i.e., there ws no evidence of restriction of phge R1-T y either host. These dt indicte tht neither the ility of the DNA to hyridize nor the EcoR digest gel electrophoresis ptterns were ltered y the growth of the temperte phge in n indictor strin. Dly nd Fitzgerld (1) similrly found tht detection of DNA homology ws unffected y growth of phge through n lternte host. However, in their study the lternte host ws restrictive nd led to modifiction of the FG. 4. () Gel electrophoresis of EcoR digests of DNA from phges R1-T nd 853 (AM1). () Autordiogrm prepred fter hyridiztion of 32P-leled DNA from phge R1-T (MC preprtion) with DNA from other preprtions of phges R1-T nd 853 (AM1). Lne 1, X; lne 2, 853 (AM1); lne 3, R1-T (MC); lne 4, R1-T (AM,); lne 5, R1-T (368). A fint nd of homology ws visile t the rrow in lne 2. phge DNA nd ltertion of the restriction endonuclese ptterns. Hyridiztion of temperte phge DNA with cteril DNA. 32P-leled DNAs from temperte phges R1-T, BK5-T, nd 134-T were hyridized to DNA from cteril strins, including their lysogenic hosts. Figure 5 shows the hyridiztion of phge BK5-T DNA with DNA from S. cremoris BK5- Three similr nds were present in DNA from the phge 1 2 4i&L _ C FG. 5. Hyridiztion of 32P-leled DNA from phge BK5-T with DNA from its lysogenic host, S. cremoris BK5. Lne 1, phge BK5-T; lne 2, S. cremoris BK5. DNA frgment A ws present in the phge DNA ut not the cteril DNA. DNA frgments B nd C were present in the cteril DNA ut not the phge DNA. All DNAs were digested with EcoR efore frctiontion.

5 + - 3T ~ F cn _- 3~ - r. ) s ' + O + - "-. + o-- - +, - W- W '.- x ō _ + ^, o" -, , --,C scjs '" s (2:, -- _ w + + -X P. -, _ >A ON _ ol -P x 'C71, 1 ' - - ' '- lī_ - _ * l H C' - -o C'f cr> ' 2 x 2_ Qg C' B cn <2 -o 1 U' CD U'. n 2 n CA l< z Gr V-~ H tz r. E o YQ ' 3 n D zl< P7 co CL ~ 5 P: zz n CD C_. P, C; Z LACTC STREPTOCOCC AND THER PHAGES 135 CO) t,q (,Q. *X -,, _~~ tt _. oo C, VOL. 47, 1984 z C 2 - -C- '3C - (ON- - 1 = w CD l- l i- i - ' ka n * X~ v: C -

6 136 JARVS -- FG. 6. Autordiogrm prepred fter hyridiztion of 32P-leled DNA from phge R-T (lne 2) with DNA from its lysogenic host, S. cremoris R (lne 3), nd phge-cured derivtive of S. cremoris R, (lne 1). DNA frgment A ws seen only in the phge DNA. DNAs were digested with EcoR efore frctiontion. (lne 1) nd its lysogenic host (lne 2). One nd in the phge DNA (A) 'ws not present in the cteril DNA. One homologous higher-mw nd (B) nd two low-mw nds (C) were seen in the cteril DNA ut not in the phge DNA. t is likely tht the nd seen in the phge ut not the cteril DNA contined the ttchment site of phge DNA to the cteril chromosome. f one ssumes similr APPL. ENVRON. MCROBOL. insertion mechnism to tht found in X phge, the points of ttchment would e present in one nd in the phge DNA ut divided etween two nds when the phge DNA hs een inserted into the cteril DNA. Hyridiztion of leled DNA from phge 134-T with DNA from its lysogenic host, S. cremoris 134 (not shown), showed tht seven of the eight nds seen in the phge DNA were present in the cteril DNA. One phge nd ws lcking in the cteril DNA, nd there were four dditionl nds in the cteril DNA not present in the phge DNA. DNA from phge R1-T to S. cremoris R, DNA (Fig. 6) showed six nds of homology in the cteril DNA (lne 3) similr in size to nds in the phge DNA (lne 2). One mjor nd (A) present in the phge DNA ws missing from the cteril DNA, nd two wek low-mw nds seen in R1-T in Fig. 2 (not shown in Fig. 6) were not seen in R, DNA. No dditionl nds were seen in the cteril DNA. To confirm tht the homology detected ws etween phge nd prophge DNA, cells of S. cremoris R1 were cured y plting on M17 gr nd exposing to UV light to produce 9% kill of cells. The pltes were then incuted t 3 C. Colonies which grew were replted nd tested for sensitivity to phge R1-T. A phge-sensitive isolte ws grown in roth, nd DNA ws extrcted from it. DNA from such n isolte is shown in lne 1 in Fig. 6. No DNA homology ws evident etween phge R1-T nd the cured isolte of S. cremoris Rl. Hyridiztion of DNA from the three temperte phges to DNA from cteril strins other thn their lysogenic hosts showed vrious degrees of homology (Tle 3). Phge R1-T DNA ws hyridized with DNA from 33 cteril strins. From 1 to 1 nds of homology were found with 23 strins. No homology ws seen with DNA from Streptococcus ,-~~~~~~~~~~P ~,, -sm :~~~~~~~~ FG. 7. () Gel electrophoresis of EcoR digests of DNA from phge R1-T nd eight cteril strins, ll of which re S. cremoris, except S. lctis ML8 nd 17 nd S. thermophilus ST33. () Autordiogrm prepred fter hyridiztion of 32P-leled DNA from phge R1-T With cteril DNA. Lne 1, 194; lne 2, 134; lne 3, 112; lne 4, phge R1-T; lne 5, 17; lne 6, 14; lne 7, ST33; lne 8, ML8; lne 9, H2.

7 VOL. 47, 1984 thermophililus ST33 or with 5 of the 12 rw milk isoltes. Representtive results re given for phge R1-T in Fig. 7. Gel electrophoresis of the EcoR digest of phge nd cteril DNA is shown in Fig. 7. The phge DNA nds re not clerly visile ecuse of the rtio of 1:1 for phge to cteril DNA. Phge 134-T DNA ws hyridized with DNA from 31 cteril strins. n generl, DNA from phges R1-T nd 134-T showed homology with the sme cteril DNAs nd lcked homology with the sme cteril DNAs (Tle 3). Phge BK5-T DNA ws hyridized with DNA from 11 cteril strins, two of which showed six nds of homology, the reminder showing no homology with the phge DNA. Hyridiztion of temperte phge DNA with plsmid DNA. Some temperte phges, such s Escherichi coli phge P, exist in the cteril cell s plsmid DNA (4). The cteril DNA used in the ove experiments would e expected to contin some plsmid DNA. To determine whether prophge DNA ws in the plsmid or chromosoml DNA for the lysogenic strins, plsmids were extrcted from 1 strins, including S. cremoris BK5, R1, nd 134. The plsmids were seprted on.8% grose gels nd trnsferred to nitrocellulose filters. One to ten plsmids were detected in these gels. Hyridiztion of leled phge DNA to plsmid DNA did not detect ny prophge DNA in the plsmids (dt not shown). Although it is possile tht not ll plsmids present in the cteril cells were isolted y the method used, these results suggest tht plsmid DNA ws not the origin of the temperte or lytic phges studied. Homology etween DNA from phges isolted from cheese plnts nd cteril DNA. 32P-leled DNA from six smll isometric phges, three prolte phges, nd two lrge isometric phges were hyridized with DNA from up to 36 strins of lctic streptococci. Little or no homology ws detected (Tle 3). The cteril strins included 19 strins which were used s strters in cheese plnts etween 1964 nd 198. The phges were isolted from cheese plnts during the sme period. These hyridiztions were crried out to determine whether the phges from cheese plnts hd originted s prophges in lysogenic strters or other strins of lysogenic lctic streptococci. To further test this, phges nd strter strins from cheese plnt opened in 1981 were exmined. Six strins of S. cremoris were in use in the plnt during the 1981 to 1982 seson. Phges were isolted from cheese wheys which grew on three of these strins (ech phge growing on only one strter strin). All phges were of the smll isometric type. DNA from ech phge ws hyridized to DNA from the six strter strins in use in the cheese plnt, using oth totl cteril DNA nd plsmid DNA. No homology ws detected etween phge nd plsmid or totl cteril DNA, except for one nd of homology etween DNA from phge 137 (588) (Fig. 8, lne 5) nd totl DNA from S. cremoris 582 (lne 1) nd S. cremoris 484 (lne 2). DSCUSSON Mny strins of lctic streptococci hve een shown y induction experiments to e lysogenic. n few instnces, the phges seen fter induction hve een propgted on other strins of lctic streptococci. Lysogenic strters nd rw milk strins must therefore e potentil source of lytic phges. However, in the present study, no evidence could e found tht the phges investigted, which hd een isolted from cheese plnts, cme directly from lysogenic strter strins or from the other strins of lctic streptococci studied. LACTC STREPTOCOCC AND THER PHAGES FG. 8. Autordiogrm prepred fter hyridiztion of 32P-leled DNA from phge 137 (588) with DNA from cteril strins. Strins underlined here were in use in the cheese plnt when phge 137 (588) ws isolted. Lne 1, 582; lne 2, 484; lne 3, 22; lne 4, 588; lne 5, phge 137 (588); lne 6, 134; lne 7, 112; lne 8, 14; lne 9, Ri; lne 1, 13; lne 11, 168; lne 12, 584. All DNAs were digested with EcoR efore frctiontion. First, the temperte phges studied showed little or no reltionship y DNA-DNA homology with phges isolted from cheese plnts, including phges of the sme morphologicl type. n contrst, phges from cheese plnts were relted to one nother if they were similr in morphology, in greement with findings reported erlier (6). Second, hyridiztion of DNA from phges isolted s lytic phges from cheese wheys showed little or no homology with cteril DNA from strter strins or from other strins of lctic streptococci. This ws prticulrly conclusive when the strter strins in use during the first seson of new cheese plnt nd phges isolted from the plnt were studied. However, prophge DNA homologous with DNA from temperte phges 134-T, R1-T, nd BK5-T ws redily detected in the DNA of their lysogenic hosts. The lck of homology etween DNA from phge R1-T nd cured derivtive of S. cremoris R1 confirmed tht the homology etween phge DNA nd cteril DNA ws specific for prophge DNA. The findings of this study re therefore in greement with the conclusions of Teuer nd Lemke (18) tht temperte phges re of minor importnce s source of phges in cheese plnts. Dt presented here contrst with those from nother recent study (14) in which DNA-DNA homology ws used to show tht virulent phge ws derived from lysogenic strin of Lctocillus csei used in lctic cid fermenttion. n tht study DNAs from the temperte nd lytic phges were identicl in restriction endonuclese pttern, nd the lytic phge ws detected in the DNA of the lysogenic cteril strin. n the present study, none of the temperte phge DNAs ws similr in restriction endonuclese pttern to ny of the lytic phges exmined. The three temperte phges studied were relted to one nother y DNA homology. Two of these, R1-T nd 134-T, were lso similr to one nother in morphology. Unexpectedly, they lso showed prtil homology with the other temperte phge, BK5-T, which differed morphologiclly in hving longer til. t hs een reported (6) tht mong 25 12

8 138 JARVS lytic lctic streptococcl phges studied, phges which differed in morphology were not relted to one nother y DNA-DNA homology under the conditions used. On the other hnd, lctic streptococcl strins used in the present study hve high degree of reltedness y DNA-DNA homology (8). This suggests tht temperte phges my hve developed from common ncestor, s the cteril strins hve developed nd differentited. This view ws supported y the high degree of homology etween DNA from temperte phges 134-T nd R1-T nd cteril DNA. Homology with their lysogenic hosts indicted the presence of prophge DNA nd ws expected. Homologous nds in lrge numer of other lctic streptococcl strins, mny of which hve een induced in previous studies to produce phges (2, (17), suggested tht reltionship exists etween these temperte phges nd other prophges resident in the cteril strins. Such reltionship ws lso suggested y the finding tht DNA from phges R1-T nd 134-T, which were relted to one nother y DNA-DNA homology, generlly formed homologous nds with DNA from the sme cteril strins. This pprent homology etween temperte phges my e relevnt to the difficulty experienced in finding indictor strins for induced phges. t is possile tht relted prophges in different lctic streptococcl strins re homoimmune with one nother, nd thus phges relesed from lysogenic strter cells cnnot propgte on other strter strins. f, on the other hnd, lytic phges isolted in cheese plnts were unrelted to temperte phges, they would not e restricted y the presence of prophges in strter cells. They therefore would e more likely to e le to ttck strter cells nd thus ecome estlished in cheese plnt. ACKNOWLEDGMENTS m indeted to H. A. Hep for isoltion of mny of the phges from cheese plnts. lso thnk C. J. Hollnd for excellent technicl ssistnce nd B. D. W. Jrvis nd R. C. Lwrence for helpful discussions. The coopertion of the Electron Microscope Unit, Deprtment of Scientific nd ndustril Reserch, Plmerston North, The X-ry Unit, Deprtment of Veterinry Clinicl Services, Mssey University, Plmerston North, nd the nstitute's Photogrphic Unit is lso grtefully cknowledged. LTERATURE CTED 1. Dly, C., nd G. F. Fitzgerld Bcteriophge DNA restriction nd the lctic streptococci, p n D. Schlessinger (ed.), Microiology Americn Society for Microiology, Wshington, D.C. 2. Hep, H. A., G. K. Y. Limsowtin, nd R. C. Lwrence Contriution of Streptococcus lctis strins in rw milk to phge APPL. ENVRON. MCROBOL. infection in commercil cheese fctories. N.Z. J. Diry Sci. Technol. 13: Huggins, A. R., nd W. E. Sndine ncidence nd properties of temperte cteriophges induced from lctic streptococci. Appl. Environ. Microiol. 33: ked, H., nd J. Tomizw Prophge Pl, n extrchromosoml repliction unit. Cold Spring Hror Symp. Qunt. Biol. 33: Jrvis, A. W The serologicl differentition of lctic streptococcl cteriophges. N.Z. J. Diry Sci. Technol. 12: Jrvis, A. W Differentition of lctic streptococcl phges into phge species y DNA-DNA homology. Appl. Environ. Microiol. 47: Jrvis, A. W., H. A. Hep, nd R. C. Lwrence The origin of cteriophges in cheese fctories. n M. W. Loutit nd J. A. R. Miles (ed.), Microil ecology. Springer-Verlg, Berlin. 8. Jrvis, A. W., nd B. D. W. Jrvis Deoxyrionucleic cid homology mong lctic streptococci. Appl. Environ. Microiol. 41: Johnson, J. L Genetic chrcteriztion, p n P. Gerhrdt (ed.), Mnul of methods for generl cteriology. Americn Society for Microiology, Wshington, D.C. 1. Klenhmmer, T. R., L. L. McKy, nd K. A. Bldwin mproved lysis of group N streptococci for isoltion nd rpid chrcteriztion of plsmid deoxyrionucleic cid. Appl. Environ. Microiol. 35: Lelnc, D. J., nd L. N. Lee Rpid screening procedure for detection of plsmids in streptococci. J. Bcteriol. 14: Lowrie, R. J Lysogenic strins of group N lctic streptococci. Appl. Microiol. 27: Reyrolle, J., M. C. Chopin, F. Letellier, nd G. Novel Lysogenic strins of lctic cid streptococci nd lytic spectr of their temperte cteriophges. Appl. Environ. Microiol. 43: Shimizu-Kdot, M., T. Skuri, nd N. Tsuchid Prophge origin of virulent phge ppering on fermenttions of Lctocillus csei S-1. Appl. Environ. Microiol. 45: Southern, E. M Detection of specific sequences mong DNA frgments seprted y gel electrophoresis. J. Mol. Biol. 98: Terzghi, B. E., nd W. E. Sndine mproved medi for lctic streptococci nd their cteriophges. Appl. Microiol. 29: Terzghi, B. E., nd W. E. Sndine Bcteriophge production following exposure of lctic streptococci to ultrviolet rdition. J. Gen. Microiol. 122: Teuer, M., nd J. Lemke The cteriophge of lctic cid cteri with emphsis on genetic spects of group N streptococci. Antonie vn Leeuwenhoek J. Microiol. Serol. 49: