Document RJv6YQVpYEJzJzEyMYMkVyjQv

14 R*prntd from Microbtologv I'JHO 19K) American Society for Microbiolo(?v Genetic Analysis of DNA-Surface Interactions in Bacillus subtilis ULDIS N. STRE1PS. SARAH HOROWITZ, a.sd RONALD J, DOYLE Deportment oj Microbiology and Immunology. School of Medicine. University of Louisville. Louisville. Kentucky 40232 In a paper which has provided the foundation for many productive experiments in molecular biology and bacterial cell division. Jacob, Bren ner. and Cuzin (11) proposed the "replicon" model to explain some of the mechanisms of DNA initiation and replication. One major con cept of their proposal was that portions of the cellular genome were attached to the cytoplasmic membrane. That assumption made it possible to account for proper segregation of the genome into daughter cells concomitant with cell growth. Subsequently, research in several laboratories demonstrated that the chromosome of various bacteria was indeed attached to the surface. Moreover, the attachment was at the origin and terminus of replication and at the replication point, as well as at several nonspecific points along the chromosome (4. 5, 9, 13, 17, 18). The specific association of the chromosome at sites involved in DNA replication reinforces the con cept of a causal relationship among DNA-surface attachment, cell division, and chromosome segregation. To study this relationship further, we have investigated DNA-surface attachments in Bacillus subtilis under conditions in which normal cell division and DNA segregation are disturbed. MEMBRANE-DNA COMPLEX IN A STABLE L-FORM A stable L-form from B. subtilis. sal-1, has been propagated in liquid media since 1969 (19). The L-form grpws in the absence of any cell wall and is stabilized by 1.2 M NaCl. sal-1 and its salt-independent derivative, sig-l. have been shown to divide aberrantly, producing progeny with altered cytoplasm and DNA contents (6, 7). In this respect, these two L-forms are similar to other L-forms isolated from B. subtilis (14). We have used sal-1 as a model for an organism which has lost proper cell division and DNA segregation control. To determine whether the DNA-membrane at tachment remained intact in these aberrantly dividing cells, we isolated DNA-membrane com plexes by use of Renografin gradients (9). The complexes were then assayed for DNA content by transformation, and membrane enrichment indices were calculated (9). As shown in Fig. I. when compared to spheroplasts (panel A), the L-form (panel B) retained enrichment for genetic markers close to the origin of replication. purAI6 and cysAl4. Several internal markers were not enriched in either sample. However, selective enrichment for genes close to the terminus of replication, such as trpC2, gltA292. citKS, and thyB, was found to be lost in the L-form but not in spheroplasts. It is attractive to speculate that this loss of replication terminus attachment con tributes to the loss of division control in the L-form. There are several factors which could have contributed to the loss of replication terminus binding (9). Among the most probable alterna tives are the absence of a cell wall or the pres ence of high salt (1.2 M NaCl) in the growth medium. If the loss of cell wall contributed to the dissociation of the terminus of replication from the surface, then it follows that the cell wall contributes to the makeup of the normal DNA-surface complex. CELL WALI^DNA COMPLEXES To examine the possibility that the cell wall is involved in attaching the chromosome to the surface of B. subtilis, we isolated cell walls at various stages of growth and examined the preparations for transforming activity (1. 15; R. J. Doyle et al., submitted for publication). Not only did isolated cell walls possess trans forming activity, but the transformation was specifically enhanced for genetic markers around the replication origin and terminus (Fig. 1C). The extent of enrichment in the cell wall differs from that observed with membrane-DNA com plexes. Thus, hisAI. pyrAI, and sacA were en riched in the cell wall samples, whereas the same markers were not enriched in membrane-DNA complexes (9, 15, 18). In contrast. cysAI4. a marker which was shown to be membraneenriched. wav not found to be specifically at tached to the cell wall (9. 15. 16). The signif icance of these differences is not obvious at the present time. There is an overall symmetry ap- 284 cma 03824 STREIPS. HOROWITZ. AND DOYLE 285 AB CD Fig. I. Generic analysis of surfnce-DNA complexes in Bacillus subtilis. Genetic markers on the chromosome of B. subtilis and its L-form, sal-1, were examined for enrichment in membrane and wall preparations (9, 15). Represented are the enrichment maps for spheroplast membrane-ONA (A). L-form fsal-IJ membrane-DNA IB), cell wall-associated DNA (C). and membrane-DNA from 1.2 M NaCI-treated cells ID). The genetic map of B. subtilis is that presented by Young and Wilson 120). Lack marker is designated as enriched (0), nonenriched IO). or not examined I*). Replication origin IO). replication terminus IT). parent in the cell wall-DNA profile (Fig. 1C), which is not present in the membrane-DNA preparations (Fig. IA and B). This loss of sym metry could be due to changes in the topography of the in vivo DNA-surface complex after re moval of the cell wall. Along with these altera tions it is possible that prolonged growth in the absence of a cell wall could also result in CMA 003825 286 DNA-SURFACE INTER \L'I IONS thtf di'MX'ucion of (ho terminu. from (he mini hr .ini. EFFECT OF SALT ON MEMBRANEDNA COMPLEXES An alternate possibility for the loss of attach ment of the replication terminus by the L-fortn is the presence of 1.2 M NaCI in the growth medium of this organism. A high concentration of salt may destroy the integrity of the surface-DNA complex. The effect of salt on morphology has been documented (3, 12). We grew B. subtilis BUL 404 imeiBIO) in 1.2 M NaCI and isolated membrane-DNA com plexes at various intervals. In Fig. ID, we show the membrane-DNA profile from cells grown in 1.2 M NaCI for 120 min. It is obvious that growth in salt dissociated the replication termi nus from the membrane in the bacterial strain. Recent experiments suggest that replication terminus dissociation occurs within the first 30 min after addition of 1.2 M NaCI to the culture growth medium. Moreover, after 120 min of growth in 1.2 M NaCI. the entire population of cells has assumed abnormal morphological char acteristics. suggestive of changes in cell division control. Thus, we have been able to emulate the L-form state of replication terminus detach ment and induce abnormal cell division by grow ing B. subtilis cells in high salt. Presumably, plasmolysis has occurred in these cells. There fore, it is attractive to postulate that the replica tion terminus site may be labile to plasmolytic detachment of the membrane from the wall. Such removal could destroy the integrity of this surface-DNA site. In contrast, the replication origin-surface complex appears to be inviolate to the effects of high salt concentrations. Origin of replication binding has also been maintained in the L-form. This suggests that the replication origin-surface complex may be of primary and vital importance for cell survival. DISCUSSION On the basis of the foregoing series of ex periments, several conclusions can be drawn concerning the surface-DNA complex in B. subtilis. First, the in vivo complex contains not only the chromosome and membrane, but also peptidoglycan. Second, of all the binding sites, the attachment of the origin of replication is of primary importance. This attachment is main tained in all samples assayed to date. Third, the replication terminus attachment appears to be labile to high salt and perhaps prolonged growth in the absence of the cell wall. Thus, the L-form and cells grown in 1.2 M NaCI have lost pi'i-tfi'eiiti.il attachment at the terminus of replication leiminu> anachment and noinial morphology can be restored after remov.il of the 1.2 M NaCI tS. Horowitz el al.. submitted for publication) Finally, once the attachment in the terminus region is lost, cells are observed to have altered morphology and division patterns. These data suggest that the DNA-surface com plex is a vital structure for the procaryotic cell and has a dynamic role in the maintenance of the cell cycle. The complex may regulate not only DNA replication but also DNa segre gation and cell division events. If the complex is disturbed, then aberrancies in cell division processes occur. It is possible that information for the proper maintenance of the complex and its functions resides in the proteins found in both the cell wall and cell membrane (2. 8, 10). Such interactions, however, remain uncharacterized at the present time. The ability to manipulate the constitution of the DNA-surface complex and the morphology of cells leads to the pos sibility that the individual functional com ponents may be isolated and studied. ACKNOWLEDGMENTS We thank Joey Dobbin* for technical avu*vtace in 4 pan of (hi* research. Terry White provided valuable (Jivcunnmmi during (he preparation of the manuscript Thu research mas supported by National Science Foundation grant PCM 79-06903 to R.J.D. and U N,S.. by a grant from the Manufacturing Chemists Association to U.N.S.. and by Institutional American Cancer Society grant IN-ltlB from (he University of LouisviUe to S H and U.N.$. LITERATURE CITED 1. Brown, W, C, R. J. D*yk. and L- N, Strripa. 1976. Comparison of various procedure* for removing pro teins and nucleic acids from cel) walls of Bdtullui mbtilis. Prep- Biochem. 4:479-466. 2. D*yk, R. J,, U. N. Sire4pa, V. & C. Fan, W. D, Bron, if. M*Mey and J. M. MansBctd. 1977. Cell wail protein tn Baalim mbnhs J Bacteriol. 129:547-549. ). ERbtt, B, Ward, and H, toprs, 1975- Formation of ceil wail polymers by reversing protoplasts of Baalim lUhtrtifttrmt*. J. Bacteriol. 124:623*632. 4. Flnialg, W. 1972 The DNA/membrane fraction of contains a DNA replication complex. J. Mol. Biol. 70:363* J97. 5. CaoesM, A. T.. aad J. Lederberg. 1965- A cell membrane- bound fraction of bacteria) DNA. Biochem. Biophys. Res. Common 16:824-635. 6. CMplii R W,, and 5, S. Nagy. 1976 Time-lapse photog raphy of Bmtlhn iuhtiln L-form*. replicating in liquid medium, i. Bactenol. L27:IOl8~IU2l, 7 Gilpin, R. W4* and S. K. PMtcmo. IV76 Adaptation of a >Ublf L-form of Rantlm *uhtili\ to minimal v^lts medium without osmotic stabilizer* i. Bacteriol. 125: K4S-H49 H Harmon, J. M-, and H. W. Tahtr. 1977 Altered ac cumulation of a membrane protein unique to a membrane-dcosynbonucleu; acid complex in a tlttu initiation mutant of Bat illm mhnii\ J, Roiicrml 130:1224-12" CMA 003826 :S6 DN VSl RF-yeS INTER V- ! IC.'S^ (he div.oRiation of the '~c Tv-1 EFFECT OF SALT ON MEMBRANED.NA COMPLEXES An alternate possibility for the loss of attach ment of the replication terminus by the L-form is the presence of 1.2 M NaCI in the growth medium of this organism. A high concentration of salt may destroy the integrity of the surface-DNA complex. The effect of salt on morphology has been documented 13. 12). We grew B. subtilis BUL 404 (meiBIO) in 1.2 M NaCI and isolated membrane-DNA com plexes at various intervals. In Fig. ID, we show the membrane-DNA prof.ie from ceils grown in 1.2 M NaCI for 120 min. It is obvious that growth in salt dissociated the replication termi nus from the membrane in the bactenal strain. Recent experiments suggest that replication terminus dissociation occurs within the first 30 min after addition of 1.2 M NaCI to the culture growth medium. Moreover, after 120 min of growth in 1.2 M NaCI. the entire population of cells has assumed abnormal morphological char acteristics, suggestive of changes in cell division control. Thus, we have been able to emulate the L-form state of replication terminus detach ment and induce abnormal cell division by grow ing B. lubtilh cells in high salt. Presumably, plasmolysis has occurred in these cells. There fore, it is attractive to postulate that the replica tion terminus site may be labile to plasmolytic detachment of the membrane from the wall. Such removal could destroy the integrity of this surface-DNA site. In contrast, the replication origin-surface complex appears to be inviolate to the effects of high sait concentrations. Origin of replication binding has also been maintained in the L-form. This suggests that the replication origin-surface complex may be of primary and vitai importance for cell survival. DISCUSSION On the basis of the foregoing series of ex periments, several conclusions can be drawn concerning the surface-DNA complex in B. subtilis, First, the in vivo complex contains not only the chromosome and membrane, but also pcptidoglycan. Second, of ail the binding sites, the attachment of the origin of replication is of primary importance. This attachment is main tained in all samples assayed to date. Third, the replication terminus attachment appears to be labile to high salt and perhaps prolonged growth in the absence of the cell wall. Thus, the L-form and cells grown in 1.2 M NaCI have N'-1 T v!.!ial 'be 1 ati.u.hrrun! and morphology Can be restored after removal ot tne I 2 M NaCI iS Horowitz el ul,. submitted foi publication!. Finally, once the attachment in the terminus region is lost, cells are observed to have altered morphology and division patterns. These data suggest that the DN.A-surt'aee com plex is a vital strueture for the priiearyotie cell and has a dynamic role in the maintenance of the cell cycle. The complex may regulate not only DNA replication but also DNA segre gation and cell division events. If the complex is disturbed, then aberrancies in cell division processes occur. It is possible that information for the proper maintenance of the complex and its functions resides in the proteins found in both the cell wall and cell membrane 12. 8. 10). Such interactions, however, remain uncharacteriaed at the present time. The ability to manipulate the constitution of the DNA-surface complex and the morphology of cells leads to the pos sibility that the individual functional com ponents may be isolated and studied. ACKNOW LEDOME.NTS We thank Joey Dobbins for techmcil avustancc m a part of this research. Terry Whitt prowded valuable divcuiviou during the preparation of the manuscript. This research w*i suoporxed by National Science Found*' lion gram PCM 7S-0490J to R.J.D- amJ U N.S.. by 4 gram from the Mamiftctunng Chemists Association to U.N.S.. and by Institutional American Cancer Society (rant IN*! MB from the University of Louisville to S.H. and U N.5. literature cited 1. Inet, W, c., ft. J. Day*. and L. N. *r*tp*. 1976. Companion of various procedures for removing pro tein and nucleic acids from cell walls of B** <//* rubntfs, Prep. Biochem. 4t479*44l. 2. Deyte, R. J., U. N. SireJp*. V. S. C. F*a, W. O. Scwwa. H. Metfcy, md J. M* .MuddS 1977. Ceil wail protein in Aaftifu1 ubiilit, J Bacterial. H9*.i47-549. J. Otatt. Tv. B. m4 tL Kipn. 1971 Formation of cell wall polymers by reverting protoplasts of &** Jltt 1 licHtmifunm*. J. BiCterwl, 124^23-632. 4, Firshalo, W. (972. The ONA/membrane fraction of pKtenmncmrrmi contains a DNA replication complex. J. Mol. Biol. TQiJFJ-397. 5 Ganemn. A. T. and J. Laderbirg. 1943- A cedi membrane- bound fraction of bacterial DNA. Biochem. Btopbyv ILcv Commun. li:S24-135. 6. GRpto, R. W.. S. S. Nat?* 1974 Time*laps* pftutug* raphy of whttU* L-forrm repBdtfmc Ifomd medium. J. Bacicriol. i27:|Otd-lU2i. 7 Gflpto, R. W.. and S. K. pKlerw, (974. Adaptation of a liable L-form of SwUImx ta/ui/iv to nummal "attv medium without omihhm; \tatnl</er> J. Bacicmd. 125: JU5,UH * Harmon. J. M., and H. W, Taber, 1977. Altered ac cumulation of a membrane pmtem unwjue (o a cm* brane-dcoxyntfc*uucJcu jckJ complex waJai uwtutkm mutant ofJ BaUerml 1345122-*-12H CMfx 003827 STREIPS. HOROWITZ. AND DOYLE 287 * Htrt'iwitt, N., R, J. IVrtl*. b V., and I. S, Strops. Sdcsiisc ,nnho in>*n \<t (he vrtr'fTn>><>/ru >ih ihc membrane m ,i huM>, l form **f H.i, </(<* w./ J bjcjcfii-i 13*41 < 10 Irruda. I.. Carroll, and V Sueftk*. I97h Mi.mhr.inv 0s* A vUrnple* m frii t!ln\ wihn/n. p llft-l'i In 0 SchlfNMnger led>. MitruNokigy --1976 Amentjn Society for Microbiology vv.ishmgmn. D C 11, Jacob, F.. ,S* flrewter, and F. Curio. 1963 Qr> the r^^ula- tion of DNA replication m biuterta Cold Spring Harbor S>mp Quant Biol 28:329-J4X, 12 kusaka, 1, 1971, Flearon microscopic obsei v.iuuns on growing and dividing protoplasts of Hmilhn meeu- frrium. J, Gen. Microbiol 63:199-202, 13 Leihowiu* P., and M. Schaectef. 1975, The attachment of the bactenaJ chromosome to the cell membrane. Int, Rev. Cytol. 41:1-28. !4. Ryur. A., and O. E. Landman. 1964. Electron micro- scope study of the relationship between mesosome loss and the stable L-sute (or protoplast state) in Baallus subttlti. i. Bactenol. 8$.*4?6-467, 13. Strtipi, U. N.t R. J. Doyle, W. D, Crabb, M. A. Courtney, and V. 5. C- Fan. 1976. Significance of DNA attachment to the ctll surface of Baaltus subnlis, p 63-68. /* A. Portolc*. R. Lopez, and M. Espinosa (ed.), Modern trends in r :r,msf 'rm.itton and lran*fi;*ti<'n t-i-c^icr \ 'r,-* r^'iiand PuNisbmv L o \m*ivrjtm in 'NutfoUa. s . K, J H*hup. S. Harford. R H. kedriU. V. 0`Sul|i*4n, and Vh O. Quinn. 1973 Lruomo'omc rcpli -aluin and *ell metabolism m frintfos wibn/n p 73S7 fn Z Vjrnfk. Z Ho<taJek. and ) Cudlm led . Genetics of indusmal microorganisms, Academia. Prague 17. Sueoka, ,N., and VI. Quinn. 1968 Membrane attachment of the chromosome replication origin n Biietllm u*hitli* Cold Spnng Harbor Symp Quant Biol, 33:69?- 705. ffl Vamagvchj. t., and H. Y&hikavi. 3977 Chromosome membrane association in fiaallut tubtilts. ill Isolation and characterisation of a DNA*proiein complex carry, mg replication origin markers. J. Mol. Biol. 110: 219-253. 19. Young* F, E., P. Haywood, aod M,, Pollock. 1970. Isolation Of L-forms of Bacillus subnhs which grow in liquid medium J, Bacterio! 102:867-870. 20. Young, F. E.. and G. A. Wilson. 1976. Revision of the linkage map of fruitful subtUis, p. 686-70], in G. 0 F&sman (ed.). Handbook of biochemistry and molecular biology. 3rd ed.. sect. B. vol. 2. CRC Press. Cleveland. Ohio. C^A 003828