JOURNAL OF BACTERIOLOGY, Mar. 1975, p. 835-847 Copyright 0 1975 American Society for Microbiology

Vol. 121, No. 3 Printed in U.S.A.

Bidirectional Chromosome Replication in Bacillus subtilis

168

NIGEL HARFORD Algemene Biologie, Irnstituut voor Moleculaire Biologie, Vrije Universiteit Brussel, B-1640 Sint-Genesius-Rode, Belgium Received for publication 18 December 1974

Density transfer analysis of deoxyribonucleic acid from Bacillus subtilis 168 thy spores germinating in 5-bromouracil medium shows the order of replication of genetic markers to be: purA16, cysA14, sacA, ctrA, (narB, arol), dal, (hisAl, purB6), (tre-12, thr-5), (argA, aroG, argC4), (metC, leu-8, pheA), (ura-1, aroD), lys-1, (trpC, metB, ilvA, citB, citK, gltA). The precise order of transfer of markers within parentheses could not be determined in these experiments. Taken together with new PBS1 transduction data presented here and in the accompanying paper of J. Lepesant-Kejzlarova, J.-A. Lepesant, J. Walle, A. Billaut, and R. Dedonder (1975), the results can be resolved in terms of a symmetric, fully bidirectional mode of chromosome replication with a replication origin close to the purA16 marker and a terminus in the region of the gltA, citK loci, diametrically opposed to the origin. A new genetic map of the B. subtilis 168 chromosome is presented.

The circular chromosome of Escherichia coli has been shown to replicate in a bidirectional fashion on the basis of evidence from gene frequency analyses (29, 7), autoradiography (34), and biochemical analysis (28). Chromosome replication data for Bacillus subtilis from both marker frequency analysis (46) and density transfer experiments (47, 31) have generally been interpreted in terms of a linear, unidirectional mode of replication although other possibilities such as bidirectionality were not excluded as an interpretation of the results

(46).

Wake (15, 38) has provided autoradiographic evidence that the B. subtilis chromosome has a circular structure and replicates in a bidirectional fashion over at least 50% of its total length. More recently, Wake has shown that during termination of replication in a temperature-sensitive mutant of strain W23, conditionally blocked for initiation of chromosome synthesis, the growing points on each arm can approach to within 10% of the total length of the chromosome (39). There is also some genetic evidence for bidirectionality. A number of sucrase markers were mapped by PBS1 transduction (25) in front of purA16, which is the earliest known marker to replicate, although one of these mutants had previously been shown to replicate after purA16 (31). Lepesant et al. (25) suggested that the discrepancy between the results from transduction mapping and density transfer could be

explained by bidirectional replication from an origin located near purA 16. A further map extension beyond the sucrase markers has been reported by Winter and Zahler (personal communication; F. E. Young and G. A. Wilson, in R. C. King (ed.), Handbook of Genetics, in press). Bidirectional replication over a limited region including the purA16 locus was later demonstrated by Hara and Yoshikawa (16) by using a number of newly isolated temperaturesensitive mutants. These workers concluded, however, that one direction of replication was blocked in the vicinity of the sacA locus shortly after initiation and that replication over the chromosome as a whole was highly asymmetric. Current map compilations for B. subtilis (10, 48; Young and Wilson, in press) are based on density transfer data from strain W23 (11) and in part on data from the genetic activity of deoxyribonucleic acid (DNA) released during strain 168 spore germination (9). These data do not agree in some respects with the map order derived by O'Sullivan and Sueoka (31) from density transfer experiments with the transformable strain 168, especially with respect to the relative map positions of an early replicating locus for histidine requirement and the ura-1 marker. A reinvestigation of chromosome mapping by density transfer analysis was begun to ascertain whether the order of marker replication in strain 168 was consistent with a uni- or bidirectional mode of chromosome replication.

835

836

J. BACTERIOL.

HARFORD MATERIALS AND METHODS

Bacterial strains. The strains of Bacillus subtilis 168 used in this study are listed in Table 1. Media. The minimal salts medium used was that of Anagnostopoulos and Spizizen (4). 5-Bromouracil (BU) medium for spore germination is Spizizen medium supplemented with 0.05% casein hydrolysate, 50 gg of tryptophan and L-alanine per ml, 10 gg of BU per ml, and 1 ,g of thymine per ml. Transformation. Cultures were grown for competence by a two-phase, step-down procedure with 0.05% and 0.01% tryptose replacing casein hydrolysate (5). For transformation of the ctrA strain QB123, 0.3% ammonium sulfate was added to all media except during the last step-down growth period for competence development. Transformants or transductants for the sacA321 and tre-12 markers were selected on modified minimal medium (33) with 0.1% sucrose or 0.2% trehalose as carbon source. Selection for narB recombinants was made on modified minimal medium with 0.2% KNOs replacing ammonium sulfate. The cysA14 marker was selected as described previously (17), the dal marker on tryptose blood agar base

medium, and the gitA1 and citBl7 markers on minimal medium. Selection for citK transformants was made on minimal medium with 0.5% sodium glutamate replacing glucose as carbon source. Transduction. Transduction with phage PBS1 was carried out essentially as described by Hoch (18) except that chloramphenicol was not added after infection of the donor culture. Colonies arising from primary selections were transferred onto the same selective medium by using sterile tooth picks and replica plated to determine the inheritance of other markers. Recombinants for lincomycin and mitomycin C resistance or sensitivity were scored on tryptose blood agar base medium containing 75 ug of lincomycin per ml, or 0.075 or 0.05 Mg of mitomycin C per ml. Spore preparation and germination. Spores of strain 168 thy were cultivated on tryptose blood agar base plates and harvested after 7 to 10 days. Spores were freed of vegetative cells and purified by treatment with lysozyme and sodium dodecyl sulfate, followed by extensive washing (5). Germination was done in BU medium at 37 C, and samples of 30 ml were taken at 100, 115, 130, and 160 min. Further replication was stopped by chilling and the addition

TABLE 1. Strains of B. subtilis Strain designation

VUB8 VUB10 VUB11 VUB14 VUB21

Genotype

VUB24

thr-5, leu-8, metB5 ura-1, purB6 trpC2, thyA, thyB thyA, thyB purA 16, leu-8, metB5, cysA 14, ery-1 hisA1, purB6, ura-I

VUB28

ura-1, leu-8, pheA1

VUB29 VUB30 QB2 QB123 QB687 QB688 CU173 CU404 CU636 BC369 SB26 SB120 WB906 WB932 JH417 GSY292 GSY250 GSY254 GSY1025 BD71 Mu3Ou2l

lin-2 lin-4 sacA321, purA16 sacA321, trpC2, ctrA I trpC2, sacA321, tre-12 hisA 1, sacA321, tre-12 trpC2, ilvA3, citK1 ilvA8, thyA, thyB trpC2, narBI hisA1, argC4, metD trpC2, metC3 trpC2, aroD120 aroI906 aroG932 trpC2, citB17

ts230 60935

trpC2, gitA I trpC2, argA2 trpC2, lys-1 trpC2, metB4, recAl hisAl, argC4, ura-1 phe-30, ura-21 trpC2, thy, dnaC230 trpC2, met, dal

[

Source

N. Sueoka Transformation of VUB24 F. Rothman Transformation of VUBI 1 See reference 17

Transformation of BD71 with DNA from Mu8u5u6 (N. Sueoka) pheAI from SB133 (J. Lederberg); leu-8 from Mu8u1 (N. Sueoka); ura-1 from SB5 (J. Lederberg); Strain constructed by several transformation steps. See reference 17 See reference 17 J. Lepesant J. Lepesant J. Lepesant J. Lepesant S. Zahler S. Zahler S. Zahler F. Young J. Lederberg J. Hoch J. Hoch J. Hoch J. Hoch J. Hoch C. Anagnostopoulos C. Anagnostopoulos C. Anagnostopoulos D. Dubnau N. Sueoka G. Bazill E. Freese

VOL. 121, 1975

BIDIRECTIONAL CHROMOSOME REPLICATION

of NaN8 to 0.02 M. The cells were washed, suspended in 2 ml of 0.01 M tris(hydroxymethyl)aminomethane, 0.001 M ethylenediaminetetraacetic acid (pH 8.0) with 1 mg of lysozyme per ml, and incubated for 45 min at 37 C before lysis with 0.1% sodium dodecyl sulfate. The crude cell lysates were analyzed by CsCl density gradient centrifugation by using a preformed gradient procedure (8) with a total volume of 6.6 ml. Gradients were centrifuged at 28,000 rpm in a Beckman model L2 centrifuge with a Spinco SW41 rotor for 40 h at 23 C. Seven-drop fractions (approximately 100 fractions) were collected in screw-cap tubes, diluted with 2 ml of SSC (0.15 M NaCl plus 0.015 M sodium citrate), and sterilized by the addition of CHCl1. Gradient fractions were assayed for transforming activity by incubating 0.1 or 0.06 ml of DNA with 1.0 ml of competent cells for 30 min at 37 C.

RESULTS Density transfer mapping. The spore of B. subtilis 168 contains a completed, resting chromosome (45) which goes through at least one round of synchronous replication during outgrowth after germination. Spores of strain 168 thy were germinated as described above in BU medium to provide a density label for the separation of replicated from unreplicated DNA by CsCl density gradient centrifugation. Gradient fractions were assayed by transformation to establish the replicative distributions for a variety of markers. The results are presented in Tables 2 to 5 and data for selected markers are shown graphically in Fig. 1 to 4. Each transformation assay in Tables 2 to 5 is designated by an R number (in the extreme left-hand column) to enable identification of those assays where the distributions of two or three markers were assayed simultaneously. In this paper "LL" refers to light-density, unreplicated DNA; "HL" refers to hybrid, replicated molecules TABLE 2. Density transfer analysis of spores germinating in BU mediuma Marker Strain EnoptExpt tested no. tested

~Total

colonies

Total transformants (%) in:

counted HL

R16 R15 R16 R15 R21 R56 R53 R42 R17 R17

VUB21 QB2 VUB21 QB2 QB123 CU636 WB906 60935 VUB24 VUB24

a Sample

purA 16 purA 16 cysA 14 sacA321 ctrA 1 narBl

aroI906 dal hisA I purB6

taken at 100 min.

1781 2117 2362 1240 5123 1806 1576 865 864 453

LL

37.6 31.5 37.1 59.6 65.5 67.1 69.9 89.6 90.9 7.7 92.3

62.4 68.4 62.9 40.4 34.5 32.9 30.1 10.4 9.1

837

TABLE 3. Density transfer analysis of spores germinating in BU medium" Expt no.

Strain

Marker tested

R8 R12 R8 R12 R39 R5 R5 R20 R37 Rl

VUB21 QB2 VUB21 QB2 60935 VUB24 VUB24 QB688 QB688 VUB8

purA16 purA16 cysA 14 sacA321 dal hisAl purB6 tre-12 tre-12 thr-5

a Sample

Total Total transformants (%) in: colonies counted HH HL LL

3489 2776 4930 2020 1413 2634 1695 4067 2520 4996

7.2 9.6 8.6 3.0 0.5 0.9 0.4 1.8 0.0 0.3

74.4 73.3 74.2 63.7 44.1 33.7 26.8 24.9 16.4 14.3

18.4 17.1 17.2 33.3 55.4 65.4 72.7 73.3 83.6 85.4

taken at 115 min.

TABLE 4. Density transfer analysis of spores germinating in BUmediuma Strain EnoptExpt

no.

R6 R6 R23 R2 R44 R48 R10 R19 R9 R10i

VUB24 VUB24 QB688 VUB8 GSY250 WB932 BD71 BC369 SB26 BD71

a Sample

Marker colonies tested ~Total

tested

hisA l purB6 tre-12 thr-5 argA2 aroG932 argC4 argC4 metC3 ura-I

~counted 3683 1571 2902 7195 4000 5938 8386 3705 3700 8121

Total transformants (%)

in:

HH

HL

LL

3.0 2.5 1.2 1.3 0.0 0.1 0.1 NT 0.0 0.0

49.9 50.3 30.7 29.5 12.1 11.1 10.7 11.2 5.3 2.1

47.1 47.2 68.1 69.2 87.9 88.8 89.2 88.8 94.7 97.9

taken at 130 min.

with one strand substituted with BU, and "HH" to twice-replicated DNA with both strands labeled with BU. The order of early marker replication established from samples taken at 100 min (Table 2), 115 min (Table 3), and 130 min (Table 4) after the start of germination is clearly purA16, cysA14, sacA, ctrAl, aroI, narB, dal, hisAl, purB6, tre-12, thr-5, as shown in Fig. 5a. This result is in agreement with the previous density transfer mapping data of O'Sullivan and Sueoka (31) and of Huang et al. (23) with respect to the purA16, sacA, hisAl, purB6, and thr-5 loci. It is not in agreement with the data for strain W23 of Dubnau et al. (11) on whose work the current map compilation is based. These authors show an inversion in the order of replication with thr-5 preceding hisAl although it is not clear from their data how this orientation was obtained.

838

HARFORD

TABLE 5. Density transfer analysis of spores germinating in 5-bromouracil mediuma Expt no.

R41 R45 R38 R43 R43 R40 R40 R43 R50 R49 R51 R47 R54 R55 R59 R54 R,58 R51 R57

Strain

Marker tested

GSY250 argA2 BD71 argC4 SB26 metC3 VUB28 leu-8 VUB28 pheA 1 Mu3Ou21 phe -30 Mu3Ou21 ura-21 VUB28 ura-1 SB120 aroD120

GSY254 lys-1 GSY292 trpC2 VUB8 metB5 CU173 JH417 JH417 CU173 CU173 GSY292 GSY292

ilvA3 citBl7 citB17 citK1 citK1 gltAl gitA 1

Total colonies counted

7096 2920 6193 6250 7598 9219 3872 8131 1257 6774 918 1926 1136 1308 1476 357 511 1019 2118

Total transformants (%) in: HH

HL

LL

8.0 7.9 5.9 6.8 5.2 5.4 3.1 2.1 2.0 1.6 0.1

49.9 47.3 48.8 47.9 46.3 48.9 33.1 34.4 34.8 28.4 20.7 18.6 18.1 19.1 21.8 18.2 16.2 15.6 15.6

42.1 44.8

0.8 0.4 0.6 0.1 0.0 0.4 0.3 0.8

45.6 46.3 48.5 45.7 63.8 63.5 63.2 70.0 79.2 80.6 81.5 80.3 78.1 81.8 83.4 84.1 83.6

aSample taken at 160 min.

The density transfer data in Tables 3 and 4 suggest that hisAl and purB6 are close together on the chromosome in terms of time of replication. If replication is unidirectional, then hisAl should be linked to purB6 or dal by PBS1 transduction, and if bidirectional, then they may be located on opposite arms of the chromosome. No linkage of hisAl to purB6 or dal could be demonstrated by transduction crosses. The correct location of hisAl on the left arm of the chromosome was found by Lepesant-KejzlarovA et al. (26, 27). Their data, together with those of Winter and Zahler (Young and Wilson, in press) establish the following linked sequence of markers: purA16, sacS, sacA, ctrAl, narAl, gtaA12, sacU, hisAl, cysB, thr-5, sacQ. This sequence is in agreement with the map order derived from the density transfer data. Transduction data establishing continuous genetic linkage of the segment aroI, narB, dal, purB6, tre-12 to the previously mapped purA 16, cysA14, lin region (17) are presented below and in the accompanying paper of LepesantKejzlarova et al. (26). The data thus unequivocally establish bidirectional replication over the early replicating region of the chromosome (Fig. 5). The last linked markers to replicate in the early region of the chromosome are thr-5 and tre-12 on the left and right arms, respectively.

J. BACTERIOL.

The next markers to replicate are argA2, aroG and argC4, followed by metC3 and leu-8, pheAl (Tables 4, 5). Unfortunately, the replication sequence in this portion of the chromosome is not clearly displayed as a sample taken at 145 min after the start of germination was lost during fractionation. However, the transfer results, taken together with PBS1 transduction data from the literature (see Table 6), establish that the argC4, metC3, ura-1 segment must replicate in this order and contemperaneously with the aroG, argA2, leu-8, pheAl, aroD sequence. This result is in reasonable agreement with the order of transfer metC3, leu-8, ura-1, phe found by O'Sullivan and Sueoka (31) but not with the map order of Dubnau et al. (11) who placed the argA, leu, phe region after the ura-1 locus. The present transfer data do not resolve the question of the orientation of the segments headed by aroG to argA and metD to argC4 with respect to the last markers to replicate in the early region of the chromosome. The approximate replication order of late markers after aroD and ura-1 is lys-1, trpC2,

metB5, ilvA3, citB17, citK1, gltAl (Table 5, Fig. 5a), although the differences in marker distribution are small for markers replicating after lys-1. The map order from PBS1 transduction data is argA, leu-8, pheAl, aroD, lys-1, trpC2, metB5, ilvA3, citK, gltA 1, citBl7 (Table 6), implying that the terminus of the chromosome is located in the region of the glt locus and that the citB locus is on the opposite chromosome arm to the trpC2 and metB5 markers as shown in Figure 5. Location of the arol to tre-12 segment. Figure 6 shows the results of a number of two-point PBS1 transduction crosses which establish linkage of the sequence of markers aroI, narB, dal, purB6, tre-12 to the previously mapped purA16 to lin region (17). This sequence is in agreement with the density transfer data, with the exception of the relative order of

aroI and narB. Since the genetic distance be-

tween these two markers is relatively small, this is not a serious discrepancy. A more detailed but essentially identical map of the lin to tre-12

region has been constructed by LepesantKejzlarova et al. (26). An independent genetic map of the region encompassing the purB6 marker has been constructed by Naumov et al. (30) by using AR9 phage transduction. This map overlaps the sequence presented in Fig. 6 and extends much further toward the replication terminus, since the gly133 marker at one extremity was found to be weakly linked (4%) to argC4. If this orienta-

tion is correct, then the mms62 and mm864

VOL. 121, 1975

BIDIRECTIONAL CHROMOSOME REPLICATION

839

C,,, 50

U

(

z

4

0

z

4

I-

cc

4 I-

Cl,,

,,

*

{,

)t

:4

FRACTION NUMBER FIG. 1. Density transfer analysis of chromosome replication by CsCl gradient centrifugation. Sample taken at 100 min after germination. The ordinate shows the percentage of total transformants in each fraction of the gradient. The hybrid, replicated (HL) DNA peak is to the left and comprises fractions 17 to 23, for the percentage distributions given in Table 2. The markers assayed were: A, cysA14; O, dal; O, sacA321; *, aroI; and A, hisAl.

J. BACTERIOL.

HARFORD

840 5I

(n z 4

0 LL

3I

C,) z 4 I-

-J

4c

0

cr

2 '1

0.

LuJ

IL I11

I I

I"

I"i

21)

22

2

26

2It

:1)

32

FRACTION NUMBER FIG. 2. Density transfer analysis of chromosome replication by CsCI gradient centrifugation. Sample taken 115 min after germination. The HH (twice-replicated) DNA peak comprises fractions 14 to 18; the HL DNA peak is fractions 19 to 24, and the unreplicated (LL) DNA peak is fractions 25 to 32, for the percentage distributions giuen in Table 3. The markers assayed were: 0, dal; A, hisA l; 0, purB6; 0, tre-12; and A, thr-5.

markers of Naumov et al. should be located close to dal and the mms96 and rec342 loci in the region of tre-12. It should be noted that the AR9 phage transduces chromosome fragments 1.2 to 1.5 times larger than those carried by phage PBS1. Linkage of recA to thyA and citB. The density transfer results imply that the argC4 to ura-l segment may be located on the opposite chromosome arm to the argA to aroD region, since these two sequences replicate simultaneously. It seemed likely that the ura-1 marker would be linked, either directly or at one remove, to the last known marker on the argA to

citB segment, which extends past the apparent terminus of replication. Other markers in these regions include the recA locus on the origin distal side of ura-1 (19) and the thyA locus which has been mapped beyond citB by Zahler and Neubauer (S. Zahler, personal communication; Young and Wilson, in press). A weak transduction linkage between the recA and argA loci has been found by Le Hegerat and Anagnostopoulos (24). This result is not likely to be correct, since argA replicates in front of the ura-l marker to which recA is linked (Table 5, Fig. 4). PBS1 transduction crosses between a recA

841

BIDIRECTIONAL CHROMOSOME REPLICATION

VOL. 121, 1975

donor strain and a citB17 recipient showed that recA recombinants (colonies sensitive to mitomycin C) could be detected among citB+ transductants with a frequency of approximately 10% (Table 7). In similar crosses with two independently isolated thymine-requiring strains as recipients, recA recombinants were also found among thy+ transductants. Linkage of recA to ura-1 was also confirmed (Table 7). These crosses have not yet been performed in the reverse direction with primary selection for recA+ transductants. Genetic linkage between the recA, thy+ and citB markers has also been found by Lepesant-Kejzlarova et al. (26). The results for the genetic crosses involving

the thymine-requiring recipients must be treated with some caution, as the phenotype found for directly selected thy+ colonies does not correspond to previous descriptions of the thyA+, thyB genotype (3, 40). The number of thy+ colonies recovered in these transductions is very low, as direct selection results in the loss of most of the potential thymine-independent transductants (40). The majority (296 out of 297) of thy+ transductants for both the VUB11 and CU404 strains, including all thy+, recA recombinants, grow when replicated onto minimal medium-agar plates supplemented with either 200 ,ug of aminopterin alone per ml (AM medium) or aminopterin plus 50 ug of thymine

....

"IL In 1--

z 4

0

IL

z 4

I-J

4 1-

0 I-I--

2 11

z w

C) cc

a. 4,

II

I

III

2

22

21

20

30

:32

FRACTION NUMBER FIG. 3. Density transfer analysis of chromosome replication by CsCI gradient centrifugation. Sample taken 130 min after germination. The HH DNA peak comprises fractions 14 to 18; the HL DNA peak is fractions 19 to 24, and the LL peak is fractions 25 to 32, for the percentage distributions given in Table 4. The markers assayed were: A, hisAl; 0, argA2; 0, argC4; 0, tre-12; and A, thr-5.

J. BACTERIOL.

HARFORD

842 I

,,

(n I-. z 4

2 0

El

LL

cn z 4

cc -j

0

2 El

z

w

C.)

w 0.

,,

III

I2

Ito

I

I2

22

21

FRACTION NUMBER chromosome 4. FIG. Density transfer analysis of replication by CsCl gradient centrifugation. Sample taken at 160 min after the start of germination. The HH peak comprises fractions 9 to 14; the HL peak is fractions 15 to 19; the LL peak is fractions. 20 to 27, for the percentage distributions given in Table 5. The markers assayed were: 0, argC4; 0, argA2; A, aroD; A, ura-1; 0, lys-l. per ml (AMT medium). One colony from strain CU404 does not grow on either AM or AMT medium and is phenotypically ilvA+, thy+. According to previous reports, combinations of the thyA and thyB loci have the following phenotypes: wild type (thyA+, thyB+), no growth on either AM or AMT medium; thymine requiring (thyA, thyB), growth on AMT medium, no growth on AM medium; thyA+, thyB, no growth on either AM or AMT medium; thyA, thyB+, growth on both AM and AMT medium (3, 40). Thus, the majority of the thy+ colonies recovered by direct selection appear to have a phenotype corresponding to a thyA, thyB+ gen-

otype. Further experiments are necessary to demonstrate that the cotransduction between recA and thy+ is in fact a linkage to the thyA locus. Initiation of replication. Hara and Yoshikawa (16) measured the order of replication for a number of temperature-sensitive loci in relation to purA16 and sacA and concluded that chromosome replication was bidirectional, but only over a short distance, with the left-hand arm being blocked in the region of sacA shortly after initiation. A more correct conclusion is simply that in the particular experimental conditions used, the sacA locus had not substan-

VOL. 121, 1975

843

BIDIRECTIONAL CHROMOSOME REPLICATION

tially replicated in the last samples examined. In their experimental system thy spores are germinated at 30 C for 3.5 h in the absence of thymine. The time of replication initiation is then precisely defined by the addition of BU or thymine to the culture. To obtain information about the symmetry of initiation of replication, a similar experiment was repeated by germinating spores of strain 168 thy for 2.5 h at 34 C in the absence of thymine. After addition of BU (10 Ag/ml) to start chromosome synthesis, samples were taken at intervals of 8, 16, and 24 min for analysis on CsCl gradients. The results show that the order of replication is purA 16, dnaC230, cysA14 with sacA still unreplicated 24 min after initiation (Table 8). The dnaC230 locus maps very close to purA16 by PBS1 transduction (1.6 map units), and it has not yet been possible to determine on which side of purA16 it is located. The nonreplication of the sacA locus is unexpected since the genetic distances measured by transduction are approximately 127 map units for purA16 to sacA (25) and 80 to 90 units for purA16 to cysA14 (17). The sacA locus would thus be expected to display some replication in the 24-min sample where 87% of the cysA14 transformants are in 0

sacA ctrA _ narB arol

hisA

dal purB6

thr5

tre

Cla roG -

-

leu

TABLE 6. Genetic map data for B. subtilis from PBS1 transduction Chromosome region

a rgC

Referencea

General map compilations

10, 48; Young and Wilson, in press 14, 17 this paper; 12, 26, 30 25, 26, 27 2, 11, 22, 35 6, 11, 20, 21, 35, 36; S. Zahler, personal communication 13, 19, 20, 21, 36, 37

purA16-lin lin-purB6-tre-12 purA16-sacQ argA-aroD aroD-thyA

argC4-recA

aThese are selected references only, which together establish continuous linkage for the regions indicated. dn

purA16

sacA

d naC cysA

argA

the hybrid peak (78% replication of cysA14). This suggests that if initiation of replication is symmetric, the origin is located between purA16 and cysA14 in the vicinity of the novAl locus. This would extend the distance for a replication fork to travel before reaching sacA to 160 transduction units versus 30 for purA 16 and 50 for cysA14 and would explain the nonreplication of the sacA locus in both sets of experiments. Hara and Yoshikawa found one temperature-sensitive mutant which replicated before

hisA cysBBd

,

dal

X- tre

sacQj

argA

a rgC

metC leu

aroDI

ura

metC

pheA aroD

lys

ura

lys

trpC2

citB

metB : ilvA -citK -

ggit

recA

ThyA

trpC citK

git citB

T

a b FIG. 5. Genetic map of Bacillus subtilis. (a) Map showing the order of time of replication for various markers from data in Tables 2-5. The map is not to scale. The order of markers enclosed within brackets cannot be determined with certainty from the density transfer data. (b) Map of the B. subtilis chromosome from both transduction and density transfer data. 0 is the origin of replication, T is the terminus, and the arrows indicate the direction of bidirectional replication. The map conforms approximately to distances derived from transduction data. Although a genetic discontinuity is shown in the mid region of each arm of the chromosome, recent transduction results (26, 30) indicate that the probable map configuration is as shown with sacQ linked to aroG and tre-12 indirectly linked to argC4.

844

J. BACTERIOL.

HARFORD lin-2

ade-33

Iin-4

arol

dal

narB

90

tre-12

purB6

52/516)

7 9 ( 7 3/349

92(17/208) 95 20/392/

79 24/ 11 aI

l

87(42/334) 32 (163/241

b46 _

s80Lo7)(

4 84 44/278s

96 (17/406)

91 (18/207)

26 _(253/341)

78 '77/ 353/

90(35/342)

40(204/ 338)

100 (o/110/

99 (5/310)

.-

98 (15/661

FIG. 6. PBS1 transduction map of the lin-tre-12 region. The map is constructed from two point crosses. The point from the selected to the unselected markers, and map distances were calculated as 100 minus the percentage of co-transfer of the two markers. The numbers in parentheses show the actual number of co-transductants found among the total colonies tested in each cross. arrows

TABLE 7. Genetic linkage of recAl by PBS1 transduction

Donor strain

Selected Recipient marker or strain phenotype

GSY1025 (metB4, JH417 recA 1)

citB+

Transductants Class

No.

Expt 1 citB+

citB+, recA Expt 2 citB+

155 10

purA 16, and their genetic map of the initiation region shows purAl6 to the left of the replication origin. Since their genetic data were derived from transformation mapping by using highmolecular-weight DNA, it is difficult to assess the distances involved in terms of the more usual transduction map units. It is also possible to interpret the present data in terms of an asymmetric initiation of replication from an origin to the left of purA16 with a delay in the initiation of chromosome synthesis on the leftward arm.

DISCUSSION A detailed reexamination of marker repliura+ 237 VUBIO ura+ cation order by density transfer mapping ura+, recA 51 shows that the B. subtilis chromosome has a CU404a thy+ 84 thy+ fully bidirectional mode of replication with an 28 thy+, recA origin close to the purA16 marker and a terthy+, ilv+ 1 VUB110 thy' 152 thy+ minus located in the region of the glt and citK thy+, recA 32 loci. This provides a genetic confirmation of the autoradiographic data of Wake (15, 38, 39) for aTotals of three experiments. extensive bidirectional replication in this bacterium. The combination of density transfer mapTABLE 8. Initiation of chromosome replication after ping and new transduction results presented thymine starvationa here and in the accompanying paper of Lepesant-KejzlarovA et al. (26) have also enabled a Distribution (%) of total transformants major revision of the B. subtilis 168 genetic map Marker 16 min 24 min 8 min presented in Figure 5. tested Genetic continuity has now been established HL LL HL LL HL LL over the origin proximal segment of the chromo47.7 52.3 75.6 24.4 88.0 12.0 purA 16 some extending from sacQ through purAI6 to dnaC230 42.2 57.8 73.2 26.8 90.0 10.0 tre-12, with complete agreement between re17.0 83.0 61.8 38.2 87.6 12.4 cysA 14 sults from density transfer and transductional sacA321 5.5 94.5 4.0 96.0 8.8 91.2 analyses. The terminal half of the chromosome is also shown as a genetically continuous sea Spores of strain 168 thy were germinated for 2.5 h at 34 C in the absence of thymine. After addition of quence, since a gap in the right-hand terminal BU (10 ug/ml) to the culture, samples were taken at arm has been closed by the finding of co-transthe times indicated, centrifuged in CsCl gradients, duction between recA and thy+ and citB. If and analyzed for marker distribution. these mapping data are correct, then the aspA citB+, recA

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locus which is linked to both recA and ura-i (20) should also be co-transducible with thyA and possibly the citB locus. Genetic discontinuities in the mid region of each arm of the chromosome are shown on the present map. The recent finding by Lepesant-Kejzlrova et al. (26) of a weak transduction linkage between sacQ and aroG and a further weak linkage between tre-12 and the catA locus, which is cotransducible with argC4, substantiates the orientation of the terminal half of the chromosome as being that shown in Fig. 5. Further independent evidence for this map configuration comes from the observation by Naumov et al. (30) of a weak linkage between argC4 and glyl33, which is the terminal marker in a linked sequence commencing with the purB6 locus. Although these weak transduction linkages must be substantiated by further data, they provide a first indication for uninterrupted genetic circularity, in support of the autoradiographic evidence of Wake (38) for a closed, circular structure of the B. subtilis chromosome. A rough estimate of the total genetic distance from PBS1 transduction data shows that both arms of the chromosome are composed of about 13 segments, each of 90 map units. As a first approximation, the chromosome thus appears to be symmetrically distributed into arms of equal length with the origin opposite the terminus. The division of a chromosome with a molecular weight of 2.0 x 109 to 2.8 x 109 (38) into 26 limit-transducing fragments would correspond to a value of 7.7 x 107 to 1.1 x 10' daltons for a region of about 90 map units. This is close to the upper limit of 1.3 x 10' for the molecular weight of the transducing DNA fragment packaged by PBS1 (40, 41). An estimate of late replication symmetry can be made from the ura-1 and aroD markers which replicate at the same time (Table 5). The genetic distance from ura-1 to citK is estimated to be about 270 transduction map units and the distance from aroD to citK is estimated at about 310 map units. These approximations are consistent with symmetric replication terminating in the glt, citK region. Autoradiographic evidence shows that the rates of travel of the growing points on each arm of the chromosome are approximately equal until at least 90% of the total chromosome has replicated (38). The genetic map for strain 168 described here differs considerably from the density transfer map for strain W23 proposed by Dubnau et al. (11), but is in essential agreement with the linear order found by O'Sullivan and Sueoka (31). Although distinct genetic differences do

845

exist between these two strains (1, 5, 48), a more detailed examination of strain W23 can probably be expected to show the same overall genetic arrangement, especially as no difference in the gross order of replication has been found by other workers (45, 46). Borenstein and Ephrati-Elizur (9) have also constructed a genetic map for B. subtilis 168 from the order of appearance of 10 markers in transforming DNA released during spore outgrowth. The sole discrepancy between this linear map and the present data is the placement of a hisA locus which is not identical to the marker used here. One possible complication in a mapping analysis by density transfer is the effect of cumulative incorporation of BU in slowing down the rate of travel of the replication fork(s) (23). Premature restart of replication resulting in the appearance of twice-replicated (HH) DNA is already evident in the second sample taken at 115 min after the start of germination (Table 3). Thus, although the replication order of markers close to the origin is clearly displayed, midregion and terminal markers appear bunched together in late samples. For this reason, no attempt has been made to calculate map positions from the present data in the absence of an independent estimate from radioactive prelabeling of the amounts of DNA replicated. Premature reinitiation of chromosome replication during density labeling with BU is a well-documented observation and is thought to result from imbalance between the relative rates of DNA and protein synthesis (44). The use of BU as density label does not affect the overall map order, since the transfer data are in agreement with PBS1 transduction mapping and with D20 to H2O density transfer experiments (31). The question of whether initiation of replication is symmetric as in E. coli (28), or asymmetric, has not yet been satisfactorily resolved, since it is not clear whether purA16 is to the right or left of the true replication origin. Further genetic and biochemical studies should give an answer to this problem. Little is known about the structural or mechanistic features of the membrane-chromosome attachment site, although work in this direction has commenced (32, 43). If initiation of replication is assumed to be asymmetric, then certain conditions may be found in which only one direction of replication is blocked. One situation where this possibility can be examined experimentally is in cells growing with prolonged generation times where the mode of replication may be sequential rather than bidirectional.

846

HAR{FORD ACKNOWLEDGMENTS

I would like to thank Raymond Hamers for his interest and support of this work and Noboru Sueoka for the first introduction to density transfer mapping. It is a pleasure to acknowledge the cooperation and helpful discussions with Jana and Jean-Antoine Lepesant which made the resolution of this work so much easier. C. Anagnostopoulos, S. Zahler, and J. Hoch generously provided essential strains. This work was supported through an ICWB contract between the Belgian Government and the Vrije Universiteit Brussel (Sint-Genesius-Rode, Belgium).

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21. 22.

1. 2. 3.

4. 5.

6.

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11. 12.

13. 14.

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18.

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