Autoradiography of the Bacillus subtilis chromosome

J. Mol. Biol. (1966) 15,435-439

Autoradiography of the Bacillus subtilis Chromosome E. S.

DENNIS AND

R. G.

WAKE

Department of Biochemistry, University of Sydney Australia (Received 29 September 1965) The DNA content of the Bacillus subtilis spore has been redetermined and found to agree with the previous estimate by Fitz-James & Young (1959). This sets an upper limit of 3·0 X 10 9 daltons (1700 JL ofthe B form) as the amount of DNA in the B. subtilis genome. By autoradiography continuous chromosomal lengths of 800 to 900 J.L are shown to occur in vegetative cells. From recent estimates of the size of the genome, these results suggest that it exists as a single chromosome, two of which are present in the spore. However, considering the fragility of the bacterial chromosome, the present results do not rule out the possibility that the B. subtilis genome exists as a much longer chromosomal structure, only one of which is present in the spore.

1. Introduction The size and structure of the Bacillus subtilis genome are of particular interest for a number of reasons. First, we know that the B. subtilis spore contains a completed genome or exact multiples of it (Oishi, Yoshikawa & Sueoka, 1964); in other words, there is no partially replicated material present. If in fact the spore contains only a single genome, as suggested by Fitz-James & Young (1959), from a knowledge of the DNA content per spore, one can arrive at a relatively precise estimate of its size. If it is assumed to exist as a single chromosome consisting of a continuous piece of DNA in the B form, one can then predict its length. Yoshikawa & Sueoka (1963a,b) have suggested that the genome of B. subtilis does exist as a single chromosome, and that when cells are grown in an unenriched medium there is just one replicating "fork" which moves in one direction along the length of the chromosome. On the other hand, Kelly & Pritchard (1965) have presented evidence which would indicate that either the genome of B. subtilis is made up of a number of chromosomes, or it is a single chromosome consisting of several independently replicating subunits. While it is not yet possible to give as complete a picture of the B. subtilis chromosome as for that of Escherichia coli (Cairns, 1963a,b), the autoradiographic results obtained so far provide a minimum estimate of its length which is particularly significant when one considers recent estimates of the size of the B. subtilis genome.

2. Materials and Methods (a) B. subtilis strains Spores were prepared from a Marmur strain, 168 6 thym -, kindly provided by Dr H. D. Mennigmann, B. subtilis Thy-I, supplied by Dr W. R. Romig, was used for the autoradiographic studies of the chromosome. 435

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E. S. DENNIS AND R. G. WAKE

(b) Preparation of spores Spores of 168 6 thym - were prepared by spreading a small volume of an overnight Penassay broth culture on to a solid potato extract medium (Oishi et al., 1964) supplemented with 10 fLg/mi. of thymine. After incubation for 5 days at 37°C, spore formation was complete. Spores were collected, treated with lysozyme, and washed with water at least 10 times to give a final preparation completely free from extraneous material. The spores were stored in water at 2°C. (c) DNA extraction and eatimation Spore DNA was extracted with either 5% trichloroacetic acid at 90°C, or 0·5 N-HCIO~ at 70°C. For the trichloroacetic acid extractions, 10 ml. spore suspension (5'4 mgjrnl.) was treated with 5% trichloroacetic acid at O°Cfor 30 min. After centrifuging, the pellet was extracted twice with 3 mi. trichloroacetic acid at 90°0 for 10 min. The combined supernatant solutions were made up to 10 mi. and the deoxypentose measured in l·O-ml. samples by the diphenylamine method (Burton, 1956) using deoxyadenosine (Sigma Chemical Co.) as the standard. The deoxypentose remaining in the pellet after 2 extractions was found to be less than 5% of the total. For the HCIO~ extractions, a 10.mI. sample of the spore suspension was washed first with 0·5 N-HOI0 4 at O°C, and then extracted twice with 3-ml. lots of 0'5 N.HCI0 4 at 70°C for 20 min. After making up to 10 ml., the deoxypentose was measured as described above. In the case of HCIO. extractions, approximately 25% of the deoxypentose remained in the pellet. However, the total in each case, supernatant DNA pellet DNA, was found to be the same within experimental error. In all, 6 independent estimations were carried out.

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(d) Spore counts

The spore suspension (5,4 mgjml.) was diluted 1 in 100 with formaldehyde-saline (0'9% NaCI-l % formaldehyde) and counts made in the Petroff-Hausser counting chamber using a phase-contrast microscope. Altogether 5 dilutions were made and a total of more than 40 large squares, each containing approximately 70 spores, was counted. (e) Preparation of autoradiographs

B. subtilis Thy-l was grown overnight by aeration in Penassay broth at 30°C. A small volume of the cells was centrifuged and diluted 1 in 10 with minimal medium (Anagnostopoulos & Spizizen, 1961) supplemented with Casamino acids (0'04 %) and thymidine (2 fLg/ml.). The diluted culture was shaken at 37°C until the cell concentration reached lOS/mI., at which stage the cells were growing exponentially. The culture was centrifuged and resuspended in the same medium containing [3H]thymidine (2 /Lg/ml., 14 elm-mole from The Radiochemical Centre, Amersham). Incubation was continued for 2 or 3 generations (2 or 3 hr) and growth stopped by diluting 1 in 10 with NET-CN-sucrose (0,1 MNaCl,O'01 M-EDTA,O'OI M-KCN, 1·5 M-sucrose,pH 8). The culture was further diluted with the same solvent to a concentration of 10 4 cells/ml., and lysozyme (Sigma Chemical Co.) added to a final concentration of 10 /Lgjml. Portions of 1·2 ml. were added to containers of similar dimensions to that described by Cairns (1962), the bottom of which consisted of a type VM Millipore filter, and allowed to dialyse for 30 min at 37°C against a solution containing 0·05 M-NaCl, 0·01 M·EDTA and 1·5 M-sucrose at pH 8. Dialysis was continued for a further 2 hr against another lot of the same solvent containing 1'0% sodium lauryl sulphate. After this the detergent was removed by dialysis against repeated changes of 0'05 M-NaCI-0'005 M-EDTA (pH 8). The containers were then turned on their sides, the membrane filter punctured and the solution allowed to drain out. After drying at 37°C, the filters were removed, fixed to microscope slides and overlaid with Kodak ARlO autorsdiographic stripping film. They were left for 2 to 3 months at 2°C in airtight, lightproof containers over CaCl:! in an atmosphere of CO2 , and then developed for 5 to 20 min in Kodak D19B developer at 20°C. The film was remounted and photographs taken under a Zeiss photomicroscope. A micrometer scale was photographed at the same magnification and measurements of chromosomal lengths made with the aid of a map measurer.

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3. Results (a) DNA content of the B. subtilis spore Fitz-James & Young (1959) reported a value of 5·0XlO- 1 6 g (3'OX109 daltons) DNA per B. subtilis spore. Kelly & Pritchard (1965) quote a value of approximately 10 1 0 daltons for the amount of DNA per genome. Because the spore DNA content would rule out this relatively larger estimate, it has been redetermined. For a preparation of clean, highly refractile spores the results shown in Table 1 were obtained.

TABLE

1

DNA content of the B. subtilis spore DNA/ml·t Sporesjml, t DNA/spore

42·5 (8,4 (5,1

± 0·5 p.g ± 0·4) X 109 ± 0,3) X 10- 1 5 g

t Quantitative estimations were carried out on a. susponsion of 1686 thym- spores containing 5·4 mg dry wt/ml.

The value of 5·1 ± 0·3 X 10 -16 g DNA per spore is in excellent agreement with the results of Fitz-James & Young (1959). B. subtilis spore DNA is double-helical (Mandel & Rowley, 1963) and if all of it exists as a continuous piece in the B form, having a mass per unit length of 1·8 X 106 daltons/flo (Langridge et al., 1960), it would have an over-all length not greater than 1700 flo. Ganesan (1963) obtained a value of 1·3 X 109 daltons for the amount of DNA in the B. subtilis genome. This would be equivalent to a strip of DNA approximately 700 fL in length and would suggest that the spore contains more than one complete genome, in contrast to the suggestion of Fitz-James & Young (1959). (b) Autoradiography of the B. subtilis chromosome All autoradiographs have been prepared using exponentially growing cells. Studies on the spore chromosome are in progress. B. subtilis is not lysed with detergent alone, so it has been neccssary to prctreat with lysozyme in order to break open the cells for autoradiography. Completely intact replicating chromosomes from Escherichia coli have only been obtained by using lysozyme without detergent (Cairns, 1963b). One attempt to use the same procedure with B. subtilis was unsuccessful because no lysis occurred. Plate I( a) shows a relatively high concentration ofpartially unravelled chromosomes. The tendency for these structures to align themselves with one another as they stick to the membrane filter while the solution drains out past them is clearly evident. This result is very similar to the earliest ones of Cairns (1962). In Plate I(b) the chromosomes have unravelled to a much greater extent and continuous lengths of at least 300 fL can be observed. The structure near the centre of the photograph shows the result of entanglement of more than one chromosome. Because of the ease with which the chromosomes break during preparation of autoradiographs and the low probability of their becoming completely unravelled,

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one has to look at large numbers of them in order to observe relatively intact structures where continuous lengths can be traced over several hundred microns. Plate II shows two such structures. In these cases the concentration of chromosomes on the Millipore filter was 1 to 10% of those in Plate I. After lysis of the cells, the procedure used here is the same as that described by Cairns (1962), and it is considered that such structures are not the result of end-to-end aggregation of smaller units. Plate II(a) shows a single chromosomal length of 930 p" measured between the two arrows. There is one discontinuity approximately 100 p, from the left-hand end of this structure, so that the true length of the intact chromosome shown here may be closer to 830 p,. In either case, a single structure would account for approximately 50% of the spore DNA content. Plate II(b) shows a chromosome of similar length. It is 800 p, long and is of particular interest because it shows what is possibly a single replicating "fork", one arm of which has been torn away. At the position marked by the cross there is an abrupt change in the number of grains per unit length by a factor of approximately two; this is what would be expected if replication were occurring at this point.

4. Discussion The DNA content of the B. subtilis spore sets an upper limit of 3·0 X 109 daltons as the amount of DNA in its genome. This is equivalent to 1700 p, of DNA in the B form. Obviously the value of 101 0 daltons suggested by Kelly & Pritchard (1965) is too high by a factor of at least three. If the B. subtilis genome contains 1·3 X 109 daltons of DNA (Ganesan, 1963) and assuming that any protein associated with the chromosome does not contribute significantly to its length, the continuous chromosomal lengths of 800 to 900 p, observed here can only be interpreted on the basis of the genome existing as a single chromosome, two of which are present in the spore. The value of 1·3 X 109 daltons as the amount of DNA in the genome would then be a slight under-estimate. Recently, MacHattie, Berns & Thomas (1965) have suggested that the nuclear DNA complement of Haemophilus influenzae is organized as a single piece of double-helical DNA about 800 p, long. Hopwood (1965) has also shown that, like E. coli, the genome of Streptomyces coelicolor exists as a single chromosome and has suggested that this may be true for bacteria in general. Considering the fragility of the bacterial chromosome, it is considered that the present results do not rule out the possibility that the B. subtilis genome exists as a single chromosome containing 3·0 X 109 daltons of DNA and having a maximum length of 1700 p,. In this case the spore would contain only one genome. The chromosomal lengths calculated from DNA contents in the present work have assumed the double helix to be in the B form. It is well to remember that bacterial DNA could adopt the A form, at least as it dries on the membrane filter (see Wilkins & Zubay, 1959) and this would result in a 25% reduction in the chromosomal lengths predicted. 3·0 X 109 daltons of DNA would then have a length of 1250 p, and the maximum length, 930 p" so far observed would account for approximately 75% of such a structure. This work has been supported by the National Health and Medical Research Council of Australia, and the N.S.W. State Cancer Council.

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PLATE II. Rapidly growing cells were labelled with [3H]thymidine for 2 generations, In (a) the chromosome is completely unravelled whereas in (b) it has crossed over itself ( x ) in one region, The scale shows 100 fL' In both Plates the arrows indicate the measured ends of the chromosomes.

THE BACILLUS SUBTILIS CHROMOSOME

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Note added in proof: The DNA content of deoxyribonuclease-treated Thy-1 spores has been estimated and found to be the same as that of 168 6 thym - spores. REFERENCES Anegnostopoulos, C. & Spizizen, J. (1961). J. Bact. 81, 741. Burton,·K. (1956). Biochem, J. 62, 315. Cairns, J. (1962). J. Mol. Biol. 4, 407. Cairns, J. (1963a). J. Mol. Biol. 6, 208. Cairns, J. (1963b). Cold Spr. Harb. Symp. Quant. Biol. 28, 43. Fitz-James, P. C. & Young, 1. E. (1959). J. Bact. 78, 743. Ganesan, A. T. (1963). Thesis, Stanford University. Hopwood, D. A. (1965). J. Mol. Biol. 12, 514. Kelly, M. S. & Pritchard, R. H. (1965). J. Bact. 89, 1314. Langridge, R., Marvin, D. A., Seeds, W. E., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F. & Hamilton, L. D. (1960). J. Mol. Biol. 2, 38. MacHattie, L. A., Berns, K. 1. & Thomas, C. A., Jr. (1965). J. Mol. Biol. 11, 648. Mandel, M. & Rowley, D. B. (1963). J. Bact. 85, 1445. Oishi, M., Yoshikawa, H. & Sueoka, N. (1964). Nature, 204, 1069. Wilkins, M. H. F. & Zubay, G. (1959). J. Biophys. Biochem, Cytol. 5, 55. Yoshikawa, H. & Sueoka, N. (1963a). Proc. Nat. Acad. Sci., Wash. 49, 559. Yoshikawa, H. & Sueoka, N. (1963b). Proc. Nat. Acad. Sci., Wash. 49,806.