Document 9JKQqppJnNavwrK1J24aw89op

R&S 116551 15 TRANSFORMATION - 1978 Proc. 4th Internat. Mtg. Bacterial Transformation 245 RESTRICTED CHROMOSOME-MEMBRANE ASSOCIATION IN A STABLE L-FORM OF BACILLUS SUBTILIS. S. Horowitz. R.J. Doyle, and U.N. Streips, Department of Microbiology and Immunology, University of Louisville, Schools of Medicine and Dentistry, Health Sciences Center, Louisville, Kentucky, 40232 (USA) ABSTRACT A stable L-form (gal-1) of Bacillus subtills BR151 was found to retain a chromosome-membrane association. The attachment of the chromosome to the membrane In the L-form is specific for the replication origin but not for the replication terminus. These results are In contrast to those obtained with the parental, cell-walled strain in which both the origin and terminus of chromosomal replication are preferentially attached, to the membrane. INTRODUCTION Attachment of the chromosome to the cytoplasmic membrane and to the cell wall has been well demonstrated in cell-walled prokaryotes and is postulated to be instrumental in DNA segregation and division control [1,2,3,4]. This attachment is specific for the origin and terminus of chromosomal replication and for the replication point [1,4,5]. In Mycoplasma the chromosome was also found to be attached to the membrane [1,6]. Studies of unstable L-forms, utilizing electron microscopy, have also revealed chromosome-membrane association [7]. Yet, the genetic specificity of the chromo somal attachment in cell wall-deficient prokaryotes has not been studied. In this report, we demonstrate that a stable Lform (sal-1) of B^. subtilis BR151, which has no cell wall polymers and divides aberrantly [8,9, R.W. Gilpin personalcommunication], retains a chromosome-membrane association. The attachment Is preferential for the region at the origin of chromosomal replication, but the specific attachment of the region close to the replication terminus has been lost. MATERIALS AND METHODS Bacterial strains. The stable L-form (sal-1) of 11. subtilis BR151 [8,9] was kindly provided by R. W. Gilpin and retains the met BIO marker. The parental strain, B^. subtilis BR151 l}-3, trpC2, metBIO, was transformed to prototrophy for R&S 116552 246 lys-3 and trpC2 with _B. subtilis W168 DNA to maintain the same auxotrophic background as the L-fora. All strains used as recipients in the transformation experiments are listed elsewhere [10]. Media and growth conditions. The L-form (sal-1) was grown in sal-1 medium [10] which contains 1.2M NaCl for stabilization, according to a modification of the technique described by Gilpin et al [9], in the presence of deoxyadenosine (200 yg/ml) and ^C-thymidine (0.1 yCi/ml, 53 mCl/mmole) for about two generations. The culture was washed In NCP buffer [11] plus 1.2M NaCl and lysed in NCF buffer by osmotic shock. The parental strain, .B. subtilis BR151 metBlO, was grown and labeled under the same conditions as the L-form but without the NaCl, washed in NCF buffer, and lysed by addition of lysozyme (0.5 mg/ml). Renografin gradients. Lysates of the L-form and its parental strain were applied onto 0-382 linear Renografin density gradients [11] and were centrifuged, fractionated and Counted according to a modification of the technique described by Ivarie -and Pene [11J- Transformation procedures. The procedures for the development of competence and -transformation were according to those described by Boylan et^ al [12]7 Dilutions of the peak fractions from the Renografin gradients served as donor DNA. RESULTS Attachment of the chromosome to the membrane. Uniformly labeled DNA from an unsheared lysate of the L-form sal-1 (Materials and Methods) forms one distinct band at the lower part of the Renografin gradient. This band corresponds to the membrane-DNA (mDNA) peak which is found in the untreated lysate of the parental strain B. subtilis BR151 metBlO (results not shown). The DNA attached to the membrane of both the Lform lysate and the lysate from the parental strain contains about 802 of the label incorporated. Shearing the L-form lysate by vortexlng for 30 seconds at setting no. 6 causes a decrease in the amount of labeled DNA which appears in the m~DNA peak and generates a slower sedimenting peak which bands at the upper part of the Renografin gradient. This new peak corres ponds to the free DNA (f-DNA) peak of the parental strain (results not shown). The distribution of the uniformly labeled DNA from the L-form sheared lysate consists of about 552 label which appears as free DNA and about 302 which appears as firmly attached membrane associated DNA. This distribution is similar to that of the uniformly labeled DNA from the sheared R&s 116553 247 lysate of the parental strain [10]. Genetic analysis of the specificity of chromosome attachment. Since the DNA Isolated in the Renografin gradients can be used In transformation assays, genetic analysis for the specificity of DMA attached to the membrane was performed, using peak fractions from the sheared lysate of the L-form and the parental strain as donor DNA. Genetic markers were tested throughout the chromosome and included markers at the origin and terminus regions of chromosomal replication as well as internal loci. The specificity of the attachment was determined by the membrane enrichment index (MX), calculated according to Sueoka and Quinn [2] using leuA8 as a standard. Several transformation experiments were performed for each marker with both the L-form and the parental strain DNA peak samples,and the average MEI was calculated. An ME1 value which equals or exceeds 1.40 was taken to indicate that the marker is enriched in the DNA attached to the membrane, hence preferentially bound. An ME1 value close to 1.00 was accepted to show no specificity for the attachment to the membrane. The results from the transformation experiments are shown in Figure 1. None of the Internal markers that we have examined has any specificity for association with the membrane in both the L-form and the parental strain. The origin region of chromosomal replication (purA!6 and cysAl4) in the L-form is preferentially attached to the membrane as is the case in the parental strain. However, the region close to the replication terminus (trpC2. thyB, citK5, gltA292, and thyA) does not appear to be attached to the membrane of the L-form although it is preferentially attached to the membrane in the parental strain. DISCUSSION This study demonstrates that the L-form, sal-1, retains a membrane-chromosome association in both sheared and unsheared lysaces. This attachment is quantitatively similar to that of the parental strain and may indicate the importance of maintaining such an association for normal cell growth. Genetic analysis for the specificity of the membranechromosome association in the L-form shows that the preferential attachment of the origin of chromosomal replication is retained. However, specificity for the attachment of the terminus region has been lost, when compared to the parental strain. This finding suggests that the two sites of attachment are different, and that the attachment of the origin is of primary importance. The Joss of association of the terminus to the membrane in the L-form could be the .-at ,,-*n3 - ,4-. * V-* I &V. -5 r * * R&S 116554 248 A. ** 1 <* o ? SO * Figure 1. Genetic analysis of m-DNA from Che L-form and Its parental strain. Genetic markers along the entire chromosome of both the L-form and its parental strain were examined for specific enrichment in membrane samples (MI) by transformation (Materials and Methods). Represented is the genetic map of the L-form sal-1 (A) and of the parental strain BR151 metBlO (B). The genetic map of Bacillus subtilis was constructed from those of Lepesant-Kejzlarova e al [13] and Young and Wilson [14]. Each marker is designated as enriched ( ) or non-enrlched (o) in m-DNA samples. Replication origin (0); Replication terminus (T). R&S 116555 249 result of one or more of the following physiological and/or genetic alterations. First of all, the loss of the cell wall may have removed an outer surface site for terminus attachment [15,16]. Secondly, a loss or alteration of a binding protein(s) which aids in the specific attachment of the chromosome to the membrane (or cell wall) could be reflected in the loss of association of the terminus region. Also,a temperate bacteriophage which inserts in this area of the chromosome [17] could participate in the binding of the genome. The L-form could have lost such a bacteriophage. Finally, the presence of a high concentration of salt (1.2M NaCl) throughout the growth cycle of the L-form could be responsible for the dissociation of specific, bound regions of the chromosome [3,9]. We are presently investigating all of these possibilities. The chromosome of subtills has been found to bind to the cell wall polymer [4,15]. It is attractive to speculate that any alteration of the integrity of the cell wall-proteinmembrane-DNA complex would result in aberrant division and DNA segregation patterns. Our observation that the L-form, sal-1, of * subtills is altered in a part of the replication complex, the attachment of the terminus of chromosome replication, provides initial support for such a speculation. It also provides opportunity for further studies on the role of the bacterial outer surface in DNA replication and segregation, and cell division. ACKNOWLEDGMENTS We wish to thank F. E. Young and R. W. Gilpin for many fruitful discussions. This work was supported by an NSF grant (PCM 78-08903) to R.J.D. and U.N.S., a grant from the Manufacturing Chemists Association to U.N.S., and an Institutional American Cancer Society grant from the University of Louisville to S.H. and U.N.S. 3 .'ft 3 l,% R&S 116556 REFERENCES 1 D.W. Smith and P.C. Hanawalt, Biochin. Biophya. Acta 1967, 149.519-531. 2 N. Sueoka and W.G. Quinn, Cold Spring Harbor Symp. Quant. Biol. 1968,33,695-705. 3 H.C. Heidrlch and W.L. Olaen, J. Cell Biol. 1975,67,444460. 4 U.N. Streips, R.J. Doyle, W.D. Crabb, H.A. Courtney and V.S.C. Fan in "Modern Trends in Bacterial Transformation and Transfection", edited by A. Portoles, R. Lopez and M. Espinosa, p. 63-68, Elsevier Press, Amsterdam, 1976. 5 R.J. Hye, M.A. O'Sullivan, K. Howard and H. Sueoka in "Microbiology-1976", edited by D. Schlessinger, p. 83-90, American Society for Microbiology, Washington, D.C., 1976. 6 D.C. Quinlan and J. Manlloff, J. Bacterlol. 1972.112.13751379. 7 A. Ryter and O.E. Landman in "Microbial Protoplasts, Spheroplasts and L-forms", edited by L.B. Guze, p. 110-123, The Williams and Wilkins Company, Baltimore, 1968. 8 F.E. Young, P. Haywood and M. Pollock, J. Bacteriol. 1970, 102.867-870. 9 R.W. Gilpin, F.E. Young and A.N. Chatterjee, J. Bacteriol. 1973.113.486-499. 10 S. Horowitz, R.J. Doyle, F.E. Young and U.N. Streips, submitted for publication. 11 R.D. Ivarie and J.J. Pene, J. Bacteriol. 1970,104.839-850. 12 R.J. Boylan, N.H. Mendelson, D. Brooks and F.E. Young, J. Bacteriol. 1972.110.281-290. 13 J. Lepesant-Kejzlarova, J.A. Lepesant, J. Walle, A. Billault and R. Dedonder, J. Bacteriol. 1975.121.823-834. 14 F.E. Young and C.A. Wilson in "Nucleic Acids II", edited by G.D. Fasman, p. 686-703, CRC Press, Cleveland, Ohio, 1976. .T 251 15 W.C. Brown, R.J. Doyle and U.N. Streips, Prep. Blochem. 1976,6,479-482. 16 R.J. Doyle, U.N, Streips, V.S.C. Fan, W.C. Brown, H. Mobley and J.M. Mansfield, J. Bacteriol. 1977,129,547-549. 17 S.A. Zahler, R.Z. Korman, R. Rosenthal and H.E. Hemphill, J. Bacteriol. 1977.129.556-558. t V** & ** 4 if $ i j*- "Tt. 1r< f R&s 116557