Vol. 140, No. 2
JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 730-733
0021-9193/79/11-0730/04$02.00/0
NOTES Completed Bacillus subtilis Nucleoid as a Doublet Structure T. McGINNESS AND R. G. WAKE*
Department of Biochemistry, University of Sydney, Sydney, N.S. W. 2006, Australia Received for publication 10 September 1979
When outgrowing spores of the temperature-sensitive dna initiation mutants of Bacillus subtilis, TsB134 and dna-i, were allowed to undergo a single round of replication by shifting to the restrictive temperature soon after its initiation, both segregating daughter nucleoids appeared as clearly defined doublet structures. The components of each doublet remained together as a discrete pair, even under conditions which resulted in the formation of deoxyribonucleic acid (DNA)less cells. A doublet nucleoid was also observed at a high frequency when TsB134 spores were allowed to germinate and grow out in the complete absence of DNA synthesis at the permissive temperature. TsB134 spores were found to contain the usual "haploid" amount of DNA. It is suggested that the doublet nucleoid reflects a folding of a single chromosome into two large domains which resolve from one another under conditions of cell extension in the absence of DNA synthesis.
The DNA (or chromosome) of bacterial cells is condensed into a structure called a nucleoid, which can be observed under the light microscope (13). Nucleoids almost certainly change their shape during the replication cycle, and it is possible that this is brought about by the folding of portions of the partially replicated chromosome into individual domains as replication proceeds (3, 4). We report here the observation of completed and unreplicating Bacillus subtilis nucleoids as clearly defined doublet structures. Although it is difficult to rule out the possibility that the doublet structure represents two chromosomes that segregate together as a pair, the data summarized below support the interpretation that it is made up of a single chromosome folded into a doublet configuration. B. subtilis spores contain only completed chromosomes (1, 12), and, on the basis of nuclear segregation studies, it has been concluded that each spore contains just one copy of the chromosome (15-17). (Evidence for two chromosomes per B. subtilis spore, in all or part of the population, has been described by some authors [14, 19].) When spores of the two temperaturesensitive dna initiation mutants of B. subtilis 168, TsB134 (11) and dna-i (18), are germinated in a defined medium at the permissive temperature (340C) and shifted to the restrictive temperature (470C) at 80 min, 80 to 90% of the spores initiate a single round of replication; ini730
tiation of a second round is blocked (2; unpublished data). The single round of replication proceeds to completion, and continued cell extension at the restrictive temperature results in the formation of a division septum between the segregated daughter nucleoids (see also references 9 and 10). Acridine orange staining (16) of such preparations from both mutants gave an unexpected result. In the majority of cases (see Table 1 for results with TsB134), each of the two segregating nucleoids appeared as a doublet structure. Examples of doublets in TsB134 are shown in Fig. 1 (the restrictive temperature in this case was only 450C, as discussed below). The doublets were more clearly defined under the microscope than the photograph would indicate (see the legend to Fig. 1). The components of the doublet became more obvious with continued incubation at the restrictive temperature, but they always remained together as a distinct pair. Thus, at 260 min (after the shift to 450C at 80 min), 100% of the nucleoids were doublet in spite of extensive cell separation and formation of DNA-less cells (40% of the total cell popula-
tion). The doublet structure persisted after a second round of replication after spore germination. Germinating TsB134 spores were transferred to 470C at a later time, 120 min. Of the chains of cells that formed by 210 min, the fractions containing one, two, three, four, and over four nu-
VOL. 140, 1979
F.~
NOTES
731
TABLE 1. Nucleoid segregation patterns in outgrowing TsB134 spores shifted to 45 or 47°C after initiation of the first round of replication' Percent of nucleoids Segregation pattern (% of each type) segregating as douTemp ("C) dd 41
45
ss
ddd
dds
14
13
8
7
[68] 9
70
47
ds
dss 6
sss
4
Other' 7
blets
19
>91
66
[25] 1
[80]
[1]
I_I
"Samples for analysis were taken at 180 min in the case of the 45°C treatment and at 200 min for 47°C; cell separation had not occurred. At least 100 outgrown spores were scored for each case. See the legend to Fig. 1 for more experimental details. b Segregation patterns: dd, two doublet nucleoids; ds, one doublet plus one single nucleoid; ddd, three doublet nucleoids, etc. Brackets indicate total percentage. 'The majority of these contained one nucleoid, generally a doublet, and presumably arose from spores which did not initiate replication by 80 min. .0. W
%
..
%46 i
'.4
*
.
C
,
I
1lbl I>
.A 4
t4 '*'
.4
* ti
..
a
4"
,
Ia z
..s f
*
,
A0
b "Oft
.4
...
FIG. 1. Doublet nucleoids in germinated and outgrowing TsB134 spores. Spores (108/ml) were germinated at 34°C in the medium described previously (2) and supplemented with thymine (20 jig/ml). At 80 min the culture was diluted into an equal volume of the same medium at 45°C. At 180 min a sample was fixed by dilution into an equal volume of 20%o Formalin. The cells were washed, fixed, and stained according to the procedure of Siccardi et al. (16) except for the use of only 0.01% acridine orange. Fluorescence microscopy was performed with a Zeiss photomicroscope fitted with an HBO 50 high-pressure mercury light source and an epi-fluorescence condenser III RS containing a filter set comprising a BP450-490 exciter filter, an FT510
chromatic beam splitter, and an LP520 barrier filter. Photography employed a 10Ox immersion objective and Kodak Ektachrome (200) film for color transparencies. The positive colored transparencies were used directly for making black-and-white (negative) prints. For photography, long exposure times (5 to 10 s) were required, and fading of the image occurred. For this reason, the photographs show less detail than could be obtained by direct observation. Most chains of cells shown here contain two or three individual cells with septa separating them. Some examples of well-resolved doublet nucleoids are indicated by an arrowhead. Also indicated are (a) a chain of two cells, each containing a doublet nucleoid; (b) a chain of three cells, each containing a nucleoid; and (c) a chain of four cells, of which two are DNA-less and two contain a doublet nucleoid. Bar, 10 ,um.
732
NOTES
cleoids were 4, 27, 21, 47, and 1%, respectively. The major four-nucleoid class is most likely the result of both first-round daughters undergoing an additional round of replication; within this class 39% of all nucleoids were doublet. The three-nucleoid class almost certainly results from just one of the first-round daughters undergoing a second round of replication. This was found to occur preferentially when germinating TsB134 spores were transferred to a slightly lower restrictive temperature, 45°C, at 80 min. Density labeling experiments have shown that initiation of second rounds proceeds in approximately 10% of the first-round daughter chromosomes under such conditions (unpublished data). Figure 1 shows some chains of cells with three nucleoids; this type comprises 25% of the total (Table 1). They are reduced to 1% when 47°C is used as the restrictive temperature, and they do not occur in dna-i, where initiation of second rounds is completely blocked at 45°C. However, it is significant that when three nucleoids occur in TsB134 at 450C they are all doublet in a significant proportion of the cases. The results described above show that a completed nucleoid can display a clearly defined doublet structure. A number of experiments have been performed in an attempt to rule out the possibility that the doublet represents two completed chromosomes that do not segregate from one another because of the restrictive conditions used. (This would require that the spore originally contained two chromosomes.) When TsB134 spores were germinated and allowed to grow out into rods at 340C for 240 min in the absence of thymine, >95% showed a doublet nucleoid. In the majority of cases a septum was also present, but it always cut off a DNA-less cell. (Resolution of the nucleoid into two components in a minor proportion of B. subtilis spores growing out in the absence of thymine has been reported previously [14].) A doublet nucleoid was again observed, but at a lower frequency (10 to 50%), with outgrowing wildtype spores when DNA replication was blocked by either thymine starvation or 6-(p-hydroxyphenylazo)-uracil. In another experiment, TsB134 spores were first allowed to complete a single cycle of replication at 470C. At 120 min the cells, 3 to 4 ,m long, contained the daughter nucleoids close to one another and not yet resolved into doublets. Upon return to 34°C at this time and after incubation for a further 80 min, the resulting chains of cells were packed with many (four to eight) nucleoids, showing that initiation and completion of new rounds of replication had proceeded at the lower temperature. When 6-(p-hydroxyphenylazo)-uracil was
J. BACTERIOL.
added at 40 min after the return to 340C, however, the chains of cells obtained after a further 80 min (25 pm long and sometimes containing DNA-less cells) showed a maximum of two nucleoids (in 91% of the cases). Each nucleoid was partitioned into a separate cell within the chain and was more diffuse and not doublet. Thus, there is no indication that incubation at 34°C, after sufficient time to allow additional initiations, permits the components of the doublets, which eventually resolve after continued incubation at 470C, to segregate from one another. It also appears that once replication is allowed to proceed part way into the second round and is then blocked, the doublet structure does not form. [When 6-(p-hydroxyphenylazo)-uracil was added at the same time as the shift back to 340C, two well-segregated doublets formed by 200 min in >80% of the cases.] In a final experiment with TsB134 spores, germination and outgrowth were allowed to proceed at 340C, and replication was blocked by the addition of 6-(p-hydroxyphenylazo)-uracil at 110, 120, and 130 min with the intention of allowing the completion of the first round of replication at 34°C in some spores, but not of a second. If the restrictive conditions in previous experiments were preventing the components of the doublets from segregating completely, one might expect to see four segregating nucleoids in some cases under these circumstances. But the maximum at 260 min was again two in 10% and 41% of the cases, when the drug was added at the earliest and latest time, respectively. TsB134 spores were found to contain the usual "haploid" amount of DNA (5.3 ± 0.5 x 1"'s g, measured as described in reference 5), which is close to that for the DNA-segregating unit of vegetative B. subtilis (6). Doublet structures were also observed when exponential cultures of TsB134 were shifted to 45 or 47°C for 230 min, but they comprised only 15% of the total. It appears that the nucleoid is more likely to adopt a doublet configuration under conditions of spore outgrowth. Doublet nucleoids have also been observed in a temperature-sensitive dna initiation mutant of Escherichia coli after shifting to the restrictive temperature (7). They were not as well defined as those found here, and no particular significance was attached to them. This is probably because the particular mutant, at the time, was thought to be defective in DNA chain elongation. The significance of the doublet structure in the completed nucleoid is not clear. But, on the assumption that it represents a single chromosome, the following suggestion is made. It is possible that the completed nucleoid contains
VOL. 140, 1979
two relatively large domains of folding, each containing one of the replicating arms of the circular chromosome. They are not normally resolved from one another, but under the conditions applied here, which permit cell extension after completion of a round of replication, there is a partial unfolding of the nucleoid to allow the two domains to move apart, with the connecting DNA anchored to the membrane through the origin and terminus regions (8). This work has beeen supported by the Australian Research Grants Committee and the University of Sydney Cancer Research Fund.
LITERATURE CITED 1. Callister, H., and R. G. Wake. 1974. Completed chromosomes in thymine-requiring Bacillus subtilis spores. J. Bacteriol. 120:579-582. 2. Callister, H., and R. G. Wake. 1977. Completion of the replication and division cycle in temperature-sensitive DNA initiation mutants of Bacillus subtilis at the nonpermissive temperature. J. Mol. Biol. 117:71-84. 3. Chai, N.-C., and K. G. Lark. 1970. Cytological studies of deoxyribonucleic acid replication in Escherichia coli 15T-: replication at slow growth rates and after a shiftup into rich medium. J. Bacteriol. 104:401-409. 4. Cooper, S., and C. E. Helmstetter. 1968. Chromosome replication and the division cycle of Escherichia coli B/r. J. Mol. Biol. 31:519-540. 5. Dennis, E. S., and R. G. Wake. 1966. Autoradiography of the Bacillus subtilis chromosome. J. Mol. Biol. 15: 435-439. 6. Eberle, H., and K. G. Lark. 1967. Chromosome replication in Bacillus subtilis cultures growing at different rates. Proc. Natl. Acad. Sci. U.S.A. 52:973-980. 7. Hirota, Y., F. Jacob, A. Ryter, G. Buttin, and T. Nakai. 1968. On the process of cellular division in Escherichia coli. 1. Asymmetrical cell division and production of deoxyribonucleic acid-less bacterial. J. Mol.
NOTES
733
Biol. 35:175-192. 8. Imada, S., L. E. Carroll, and N. Sueoka. 1975. Membrane-DNA complex in Bacillus subtilis, p. 187-200. In M. Goulian, P. Hanawalt, and C. F. Fox (ed.), DNA synthesis and its regulation. W. A. Benjamin, Inc., Menlo Park, Calif. 9. Mendelson, N. H. 1968. Nuclear segregation without DNA replication in Bacillus subtilis. Biochim. Biophys. Acta 190:132-138. 10. Mendelson, N. H. 1968. Can defective segregation prevent initiation? Cold Spring Harbor Symp. Quant. Biol. 33:313-316. 11. Mendelson, N. H., and J. D. Gross. 1967. Characterization of a temperature-sensitive mutant of Bacillus subtilis defective in deoxyribonucleic acid replication. J. Bacteriol. 94:1603-1608. 12. Oishi, M., H. Yoshikawa, and N. Sueoka. 1964. Synchronous and dichotomous replication of the Bacillus subtilis chromosome during spore germination. Nature (London) 204:1069-1073. 13. Pettijohn, D. E. 1976. Prokaryotic DNA in nucleoid structure. Crit. Rev. Biochem. 4:175-202. 14. Ryter, A. 1973. Etude autoradiographique de l'etat de replication des noyaux du sporange et de la spore chez Bacillus subtilis. Ann Microbiol. 124B:3-9. 15. Ryter, A., and F. Jacob. 1966. Segregation des noyaux chez Bacillus subtilis au cours de la germination de spores. C. R. Acad. Sci. 263:1176-1179. 16. Siccardi, A. G., A. Galizzi, G. Mazza, A. Clivio, and A. M. Albertini. 1975. Synchronous germination and outgrowth of fractionated Bacillus subtilis spores: tool for the analysis of differentiation and division of bacterial cells. J. Bacteriol. 121:13-19. 17. Wake, R. G. 1976. Segregation of Bacillus subtilis chromosomes radioactively labeled during the first round of replication after germination of spores. J. Bacteriol. 127:433-439. 18. White, K., and N. Sueoka. 1973. Temperature sensitive DNA synthesis mutants of Bacillus subtilis. Genetics 73:185-214. 19. Yoshikawa, H. 1968. Chromosomes in Bacillus subtilis spores and their segregation during germination. J. Bacteriol. 95:2282-2292.
JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 730-733
0021-9193/79/11-0730/04$02.00/0
NOTES Completed Bacillus subtilis Nucleoid as a Doublet Structure T. McGINNESS AND R. G. WAKE*
Department of Biochemistry, University of Sydney, Sydney, N.S. W. 2006, Australia Received for publication 10 September 1979
When outgrowing spores of the temperature-sensitive dna initiation mutants of Bacillus subtilis, TsB134 and dna-i, were allowed to undergo a single round of replication by shifting to the restrictive temperature soon after its initiation, both segregating daughter nucleoids appeared as clearly defined doublet structures. The components of each doublet remained together as a discrete pair, even under conditions which resulted in the formation of deoxyribonucleic acid (DNA)less cells. A doublet nucleoid was also observed at a high frequency when TsB134 spores were allowed to germinate and grow out in the complete absence of DNA synthesis at the permissive temperature. TsB134 spores were found to contain the usual "haploid" amount of DNA. It is suggested that the doublet nucleoid reflects a folding of a single chromosome into two large domains which resolve from one another under conditions of cell extension in the absence of DNA synthesis.
The DNA (or chromosome) of bacterial cells is condensed into a structure called a nucleoid, which can be observed under the light microscope (13). Nucleoids almost certainly change their shape during the replication cycle, and it is possible that this is brought about by the folding of portions of the partially replicated chromosome into individual domains as replication proceeds (3, 4). We report here the observation of completed and unreplicating Bacillus subtilis nucleoids as clearly defined doublet structures. Although it is difficult to rule out the possibility that the doublet structure represents two chromosomes that segregate together as a pair, the data summarized below support the interpretation that it is made up of a single chromosome folded into a doublet configuration. B. subtilis spores contain only completed chromosomes (1, 12), and, on the basis of nuclear segregation studies, it has been concluded that each spore contains just one copy of the chromosome (15-17). (Evidence for two chromosomes per B. subtilis spore, in all or part of the population, has been described by some authors [14, 19].) When spores of the two temperaturesensitive dna initiation mutants of B. subtilis 168, TsB134 (11) and dna-i (18), are germinated in a defined medium at the permissive temperature (340C) and shifted to the restrictive temperature (470C) at 80 min, 80 to 90% of the spores initiate a single round of replication; ini730
tiation of a second round is blocked (2; unpublished data). The single round of replication proceeds to completion, and continued cell extension at the restrictive temperature results in the formation of a division septum between the segregated daughter nucleoids (see also references 9 and 10). Acridine orange staining (16) of such preparations from both mutants gave an unexpected result. In the majority of cases (see Table 1 for results with TsB134), each of the two segregating nucleoids appeared as a doublet structure. Examples of doublets in TsB134 are shown in Fig. 1 (the restrictive temperature in this case was only 450C, as discussed below). The doublets were more clearly defined under the microscope than the photograph would indicate (see the legend to Fig. 1). The components of the doublet became more obvious with continued incubation at the restrictive temperature, but they always remained together as a distinct pair. Thus, at 260 min (after the shift to 450C at 80 min), 100% of the nucleoids were doublet in spite of extensive cell separation and formation of DNA-less cells (40% of the total cell popula-
tion). The doublet structure persisted after a second round of replication after spore germination. Germinating TsB134 spores were transferred to 470C at a later time, 120 min. Of the chains of cells that formed by 210 min, the fractions containing one, two, three, four, and over four nu-
VOL. 140, 1979
F.~
NOTES
731
TABLE 1. Nucleoid segregation patterns in outgrowing TsB134 spores shifted to 45 or 47°C after initiation of the first round of replication' Percent of nucleoids Segregation pattern (% of each type) segregating as douTemp ("C) dd 41
45
ss
ddd
dds
14
13
8
7
[68] 9
70
47
ds
dss 6
sss
4
Other' 7
blets
19
>91
66
[25] 1
[80]
[1]
I_I
"Samples for analysis were taken at 180 min in the case of the 45°C treatment and at 200 min for 47°C; cell separation had not occurred. At least 100 outgrown spores were scored for each case. See the legend to Fig. 1 for more experimental details. b Segregation patterns: dd, two doublet nucleoids; ds, one doublet plus one single nucleoid; ddd, three doublet nucleoids, etc. Brackets indicate total percentage. 'The majority of these contained one nucleoid, generally a doublet, and presumably arose from spores which did not initiate replication by 80 min. .0. W
%
..
%46 i
'.4
*
.
C
,
I
1lbl I>
.A 4
t4 '*'
.4
* ti
..
a
4"
,
Ia z
..s f
*
,
A0
b "Oft
.4
...
FIG. 1. Doublet nucleoids in germinated and outgrowing TsB134 spores. Spores (108/ml) were germinated at 34°C in the medium described previously (2) and supplemented with thymine (20 jig/ml). At 80 min the culture was diluted into an equal volume of the same medium at 45°C. At 180 min a sample was fixed by dilution into an equal volume of 20%o Formalin. The cells were washed, fixed, and stained according to the procedure of Siccardi et al. (16) except for the use of only 0.01% acridine orange. Fluorescence microscopy was performed with a Zeiss photomicroscope fitted with an HBO 50 high-pressure mercury light source and an epi-fluorescence condenser III RS containing a filter set comprising a BP450-490 exciter filter, an FT510
chromatic beam splitter, and an LP520 barrier filter. Photography employed a 10Ox immersion objective and Kodak Ektachrome (200) film for color transparencies. The positive colored transparencies were used directly for making black-and-white (negative) prints. For photography, long exposure times (5 to 10 s) were required, and fading of the image occurred. For this reason, the photographs show less detail than could be obtained by direct observation. Most chains of cells shown here contain two or three individual cells with septa separating them. Some examples of well-resolved doublet nucleoids are indicated by an arrowhead. Also indicated are (a) a chain of two cells, each containing a doublet nucleoid; (b) a chain of three cells, each containing a nucleoid; and (c) a chain of four cells, of which two are DNA-less and two contain a doublet nucleoid. Bar, 10 ,um.
732
NOTES
cleoids were 4, 27, 21, 47, and 1%, respectively. The major four-nucleoid class is most likely the result of both first-round daughters undergoing an additional round of replication; within this class 39% of all nucleoids were doublet. The three-nucleoid class almost certainly results from just one of the first-round daughters undergoing a second round of replication. This was found to occur preferentially when germinating TsB134 spores were transferred to a slightly lower restrictive temperature, 45°C, at 80 min. Density labeling experiments have shown that initiation of second rounds proceeds in approximately 10% of the first-round daughter chromosomes under such conditions (unpublished data). Figure 1 shows some chains of cells with three nucleoids; this type comprises 25% of the total (Table 1). They are reduced to 1% when 47°C is used as the restrictive temperature, and they do not occur in dna-i, where initiation of second rounds is completely blocked at 45°C. However, it is significant that when three nucleoids occur in TsB134 at 450C they are all doublet in a significant proportion of the cases. The results described above show that a completed nucleoid can display a clearly defined doublet structure. A number of experiments have been performed in an attempt to rule out the possibility that the doublet represents two completed chromosomes that do not segregate from one another because of the restrictive conditions used. (This would require that the spore originally contained two chromosomes.) When TsB134 spores were germinated and allowed to grow out into rods at 340C for 240 min in the absence of thymine, >95% showed a doublet nucleoid. In the majority of cases a septum was also present, but it always cut off a DNA-less cell. (Resolution of the nucleoid into two components in a minor proportion of B. subtilis spores growing out in the absence of thymine has been reported previously [14].) A doublet nucleoid was again observed, but at a lower frequency (10 to 50%), with outgrowing wildtype spores when DNA replication was blocked by either thymine starvation or 6-(p-hydroxyphenylazo)-uracil. In another experiment, TsB134 spores were first allowed to complete a single cycle of replication at 470C. At 120 min the cells, 3 to 4 ,m long, contained the daughter nucleoids close to one another and not yet resolved into doublets. Upon return to 34°C at this time and after incubation for a further 80 min, the resulting chains of cells were packed with many (four to eight) nucleoids, showing that initiation and completion of new rounds of replication had proceeded at the lower temperature. When 6-(p-hydroxyphenylazo)-uracil was
J. BACTERIOL.
added at 40 min after the return to 340C, however, the chains of cells obtained after a further 80 min (25 pm long and sometimes containing DNA-less cells) showed a maximum of two nucleoids (in 91% of the cases). Each nucleoid was partitioned into a separate cell within the chain and was more diffuse and not doublet. Thus, there is no indication that incubation at 34°C, after sufficient time to allow additional initiations, permits the components of the doublets, which eventually resolve after continued incubation at 470C, to segregate from one another. It also appears that once replication is allowed to proceed part way into the second round and is then blocked, the doublet structure does not form. [When 6-(p-hydroxyphenylazo)-uracil was added at the same time as the shift back to 340C, two well-segregated doublets formed by 200 min in >80% of the cases.] In a final experiment with TsB134 spores, germination and outgrowth were allowed to proceed at 340C, and replication was blocked by the addition of 6-(p-hydroxyphenylazo)-uracil at 110, 120, and 130 min with the intention of allowing the completion of the first round of replication at 34°C in some spores, but not of a second. If the restrictive conditions in previous experiments were preventing the components of the doublets from segregating completely, one might expect to see four segregating nucleoids in some cases under these circumstances. But the maximum at 260 min was again two in 10% and 41% of the cases, when the drug was added at the earliest and latest time, respectively. TsB134 spores were found to contain the usual "haploid" amount of DNA (5.3 ± 0.5 x 1"'s g, measured as described in reference 5), which is close to that for the DNA-segregating unit of vegetative B. subtilis (6). Doublet structures were also observed when exponential cultures of TsB134 were shifted to 45 or 47°C for 230 min, but they comprised only 15% of the total. It appears that the nucleoid is more likely to adopt a doublet configuration under conditions of spore outgrowth. Doublet nucleoids have also been observed in a temperature-sensitive dna initiation mutant of Escherichia coli after shifting to the restrictive temperature (7). They were not as well defined as those found here, and no particular significance was attached to them. This is probably because the particular mutant, at the time, was thought to be defective in DNA chain elongation. The significance of the doublet structure in the completed nucleoid is not clear. But, on the assumption that it represents a single chromosome, the following suggestion is made. It is possible that the completed nucleoid contains
VOL. 140, 1979
two relatively large domains of folding, each containing one of the replicating arms of the circular chromosome. They are not normally resolved from one another, but under the conditions applied here, which permit cell extension after completion of a round of replication, there is a partial unfolding of the nucleoid to allow the two domains to move apart, with the connecting DNA anchored to the membrane through the origin and terminus regions (8). This work has beeen supported by the Australian Research Grants Committee and the University of Sydney Cancer Research Fund.
LITERATURE CITED 1. Callister, H., and R. G. Wake. 1974. Completed chromosomes in thymine-requiring Bacillus subtilis spores. J. Bacteriol. 120:579-582. 2. Callister, H., and R. G. Wake. 1977. Completion of the replication and division cycle in temperature-sensitive DNA initiation mutants of Bacillus subtilis at the nonpermissive temperature. J. Mol. Biol. 117:71-84. 3. Chai, N.-C., and K. G. Lark. 1970. Cytological studies of deoxyribonucleic acid replication in Escherichia coli 15T-: replication at slow growth rates and after a shiftup into rich medium. J. Bacteriol. 104:401-409. 4. Cooper, S., and C. E. Helmstetter. 1968. Chromosome replication and the division cycle of Escherichia coli B/r. J. Mol. Biol. 31:519-540. 5. Dennis, E. S., and R. G. Wake. 1966. Autoradiography of the Bacillus subtilis chromosome. J. Mol. Biol. 15: 435-439. 6. Eberle, H., and K. G. Lark. 1967. Chromosome replication in Bacillus subtilis cultures growing at different rates. Proc. Natl. Acad. Sci. U.S.A. 52:973-980. 7. Hirota, Y., F. Jacob, A. Ryter, G. Buttin, and T. Nakai. 1968. On the process of cellular division in Escherichia coli. 1. Asymmetrical cell division and production of deoxyribonucleic acid-less bacterial. J. Mol.
NOTES
733
Biol. 35:175-192. 8. Imada, S., L. E. Carroll, and N. Sueoka. 1975. Membrane-DNA complex in Bacillus subtilis, p. 187-200. In M. Goulian, P. Hanawalt, and C. F. Fox (ed.), DNA synthesis and its regulation. W. A. Benjamin, Inc., Menlo Park, Calif. 9. Mendelson, N. H. 1968. Nuclear segregation without DNA replication in Bacillus subtilis. Biochim. Biophys. Acta 190:132-138. 10. Mendelson, N. H. 1968. Can defective segregation prevent initiation? Cold Spring Harbor Symp. Quant. Biol. 33:313-316. 11. Mendelson, N. H., and J. D. Gross. 1967. Characterization of a temperature-sensitive mutant of Bacillus subtilis defective in deoxyribonucleic acid replication. J. Bacteriol. 94:1603-1608. 12. Oishi, M., H. Yoshikawa, and N. Sueoka. 1964. Synchronous and dichotomous replication of the Bacillus subtilis chromosome during spore germination. Nature (London) 204:1069-1073. 13. Pettijohn, D. E. 1976. Prokaryotic DNA in nucleoid structure. Crit. Rev. Biochem. 4:175-202. 14. Ryter, A. 1973. Etude autoradiographique de l'etat de replication des noyaux du sporange et de la spore chez Bacillus subtilis. Ann Microbiol. 124B:3-9. 15. Ryter, A., and F. Jacob. 1966. Segregation des noyaux chez Bacillus subtilis au cours de la germination de spores. C. R. Acad. Sci. 263:1176-1179. 16. Siccardi, A. G., A. Galizzi, G. Mazza, A. Clivio, and A. M. Albertini. 1975. Synchronous germination and outgrowth of fractionated Bacillus subtilis spores: tool for the analysis of differentiation and division of bacterial cells. J. Bacteriol. 121:13-19. 17. Wake, R. G. 1976. Segregation of Bacillus subtilis chromosomes radioactively labeled during the first round of replication after germination of spores. J. Bacteriol. 127:433-439. 18. White, K., and N. Sueoka. 1973. Temperature sensitive DNA synthesis mutants of Bacillus subtilis. Genetics 73:185-214. 19. Yoshikawa, H. 1968. Chromosomes in Bacillus subtilis spores and their segregation during germination. J. Bacteriol. 95:2282-2292.
