Differences in the Genetic Structure ofBacillus subtilis Strains Carrying ...

JouRNAL OF BACTERIOLOGY, May 1976, p. 609-618 Copyright © 1976 American Society for Microbiology

Vol. 126, No. 2 Printed in U.S A.

Differences in the Genetic Structure of Bacillus subtilis Strains Carrying the trpE26 Mutation and Strain 168 J. TROWSDALE' AND C. ANAGNOSTOPOULOS* Centre de Ggn&tique Mok1culaire, C.N.R.S., 91190-Gif-sur-Yvette, France

Received for publication 12 December 1975

It was previously shown that in strains ofBacillus subtilis bearing the trpE26 mutation a chromosome segment (from trpD to ilvA) is translocated to a position near the thr region. Further PBS1-mediated transduction data have now revealed that these strains also possess an inversion of part of the chromosome from the origin of replication, down to the tre locus on one side and the cysB locus on the other. These data concern evidence of linkage of tre-12 to markers in the translocation (hisH2, tyrAl, and metB3) as well as linkage of the cysB3 marker to thi-86, gly-133, and catA. They explain the previously observed absence of linkage of markers in the translocated segment to cysB3. The model proposed for the formation of merodiploids in trpE26 strains, which calls for the fusion of two genetic elements, is not incompatible with this new finding. Moreover, the model was found to be of more general significance. Evidence is presented that merodiploids may also be produced from 168 recipients and trpE26 donors by transduction, when selection is made for particular markers. The markers should be in the ilvA and thr regions. They are widely separated in the recipient 168 strain but are closely linked in the trpE26 donor as a result of the translocation. The diploids so produced are unstable, sensitive to L-lysine, and segregate rapidly.

Strains of Bacillus subtilis carrying the trpE26 mutation possess certain properties which distinguish them from those of 168 origin (1, 2, 3). The most important of these features is their aptitude to give rise to merodiploid clones when transformed or transduced to tryptophan independence. In a recent paper we have shown that the trpE26 marker is the result of the translocation of a chromosome segment (12). The relevent evidence was obtained by studies on the replication order of markers, using the density transfer technique, and from the results of mapping experiments. Markers on a chromosome segment extending from trpD to ilvA replicated earlier in trpE26 strains, at about the same time as tre-12 and thr-5. These two markers (the replication times of which are close in strain 168) are situated on opposite sides of the bidirectionally replicating chromosome (5, 9, 14). Those markers normally flanking the region (trpE8 and citK7) replicated late, as usual. The results suggested that the trpD-ilvA segment was shifted in strains bearing the trpE26 marker to a position closer to the origin of replication, near tre or thr. Mapping experiments by PBS1 transduction, when both donor

and recipient carried the trpE26 marker, clearly showed that the segment was removed from its normal position to a site near thr (i) aroB2 and citK7 were linked in these strains; (ii) linkage of cysB3 and thr-5 was lost; (iii) several markers in the translocation were linked to thr-5 and ald. However, similar transduction crosses failed to reveal linkage of the translocation to cysB3. These negative results have now been explained by the results of the mapping experiments reported in the present paper. They show that in strains carrying the trpE26 mutation the chromosomal rearrangements are more extensive. Not only is the trpD-ilvA segment translocated to a position near thr, but the whole of the origin-proximal region of the chromosome down to cysB on one side, and tre on the other, is inverted with respect to the terminus of replication. These results make little difference to the previously proposed model for the formation of merodiploids when strains carrying the trpE26 mutation are transduced or transformed to Trp+ (12). This model was based on the fact that the trpE gene is split into two parts in the recipient strain; the portion of the gene normally further away from the origin is now closest since it forns one end point of the translocation. Hence, trpE26+ recombinants are formed

I Present address: Department of Microbiology, Scripps Clinic and Research Foundation, La Jolla, Calif. 92037.

609

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J. BACTERIOL.

TROWSDALE AND ANAGNOSTOPOULOS

by the fusion of two genetic elements (two chromosomes in a binucleate cell or two arms of a chromosome in the course of replication) which produces a chromosome with a long tandem duplication. We have recently obtained results that indicate that the model may be of more general significance. As will be shown merodiploids are also formed when recipients of strain 168 genetic background are transduced by trpE26 donors, if selection is made for particular genetic markers. MATERIALS AND METHODS Media and procedures for PBS1 transduction have been described in previous papers (2, 7, 12). Bacterial strains. The B. subtilis strains used are listed in Table 1. In the text the terms "trpE26 strain" and "168 strain" will be used respectively to designate strains carrying the trpE26 mutation and strains of the normal chromosome structure of B. subtilis 168. Scoring of phenotypes of transductants. For auxotrophic markers amino acids were added to minimal plus glucose medium (MG) plates at a concentration of 20 ,ug/ml. The glucose in MG was replaced by 1 rmg of trehalose/ml to test the Tre phenotype. To test the sensitivity of transductants to L-lysine (sensitive, Lys"'n; resistant, Lysres) a concentration of 164 ,ug of L-lysine/ml of MG was used. The thi-86 marker confers a requirement for thiamine (1 ,ug/ ml) at 37 C but not at 30 C (13). hom-i clones require methionine or homoserine, plus threonine (Leibovici and Anagnostopoulos, unpublished data). nov-3 confers resistance to 2 ,ug of novobiocin/ml in tryptose blood agar base (Difco) plates. The phenotype of catA strains (Prth, high production of extracellular proteases in the presence of glucose) was scored as the presence of halos around the colonies on sporulation medium plates containing 1.5% heatdenatured bovine serum albumin and 2% glucose. The plates were incubated for 36 to 40 h at 37 C (9). RESULTS Genetic map of trpE26 strains. The results ofthe PBS1 transduction crosses reported in the

previous paper have clearly positioned the translocated segment (trpD-ilvA) close to the thr region and more proximal to the origin in trpE26 strains (12). The absence of linkage of markers in the translocation to cysB3 was therefore puzzling. The replication time of thr-5 in 168 strains is essentially the same as that of tre-12, a marker situated on the opposite half of the chromosome (14). We considered the possibility that the translocated segment was linked to this marker. The tre-12 marker was introduced into a trpE26 strain and this strain was used as donor or recipient in PBS1 transduction crosses, the other partners being strains possessing various markers (hisH2, tyrAl, metB3) in the translocation as well as trpE26. These

experiments revealed a rather strong linkage of tre-12 to all markers tested of the trpD-ilvA segment. The data are presented in Table 2. In this table are also included some new results on the linkage of the translocation to other markers on the thr side (nov-3 and hom-i). The map of this chromosome region in trpE26 TABLE 1. Strains of B. subtilis used No. WT 166 168M VUB33

QB804 QB858 1486r

GSY273 GSY293 GSY343 GSY378 GSY468d GSY1201 GSY1210 GSY1216 GSY1269 GSY1763 GSY1971 GSY1972 GSY1973 GSY1977 GSY1988 GSY1992 GSY1997 GSY1999 GSY2000 GSY2204

Genotype, Wild-type phenotype trpE26 trpC2 trpC2 nov-3 thyAl thyBI hisAl catA pha-1 metDI gly-133 sacA321 trpC2. thi-86 (GezALA86ts phenotype) ilvAl trpC2 hom-1 thr-5 aroB2 ilvCl trpE26 ilvCl trpE26 metB3 trpE26 ilvA2 trpE26 tyrAl hisH2 trpE26 ilvCl

trpE26 hom-i

trpE26 cysB3 trpE26 thr-5 trpE26 hisH2 trpE26 nov-3 hom-i ilvAl trpE26 tre-12 trpE26 argC4 trpE26 catA trpE26 metDl trpE26 gly-133 metDI trpE26 thi-86 trpE26 gly-133 trpE26 tre-12 hisH2 trpE26 metDI cysB3

Originb Burkholder and Giles (4) Burkholder and Giles (4) N. Harford J.-A. Lepesant J.-A. Lepesant

Trowsdale and Smith (13)

166

GSY378eJ

166 GSY378eef GSY293 -* GSY468e

VUB33 -. GSY19729 GSY273 -. GSY2939

QB804 GSY19979 QB858 - GSY19979 QB858 - GSY19979 1486 -. GSY19929.h GSY2205 GSY2206 GSY1971 - GSY22049 GSY2207 GSY1992 GSY12169 GSY1971 GSY22049 GSY2209 GSY2214 trpE26 cysB3 gly-133 GSY2206 GSY2209e a See (14) and Materials and Methods for a description of the genetic markers. b"GSY" strains were isolated in this laboratory. Those strains for which the origin is not given are from the laboratory stocks and have been described previously. I In strain 1486 the designation thi (ts) was previously given to the thi-86 marker (13). This marker was about 40% cotransducible with tre-12 in 168 strains (unpublished data) so it is probably in the thiA locus (14). GezALA86ts is a germination marker present in strain 1486 not used in this study. d All the trpE26 strains, other than strains 166 and GSY1269, have the genetic background of strain GSY468. e Strains obtained by transformation. The arrow points to the recipient strain. f Strains obtained by identical crosses but in different experiments. o Strains obtained by transduction. The arrow points to the recipient strain. hThe thi-86 marker was introduced into strain GSY1992 at low frequency by transduction to Tre+, although in trpE26 strains thi-86 and tre-12 are on opposite sides of the chromosome (see Discussion).

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GENETIC STRUCTURE OF B. SUBTILIS STRAINS

strains constructed from these results as well as those of the previous paper is given in Fig. 1. It was now evident that in trpE26 strains the translocated segment is linked on one side to tre and on the other to thr. The fate of the cysB region (linked to thr in strain 168), and the glycatA region (linked to tre in strain 168) remained to be determined. From a consideration of the B. subtilis 168 genetic map (14) it seemed

611

likely that these two regions would now be linked to each other. This was readily demonstrated by means of a series of PBS1 transduction crosses, shown on Table 3. Both catA and gly-133 were cotransduced with cysB3 at a low but significant frequency and had the known linkage relationships to markers on their right hand side (metDl and argC4). Further evidence of linkage of cysB3 to this region was

TABLE 2. Linkage relationships of markers in the tre-thr region of the chromosome of trpE26 strains measured in PBS1 transduction experiments" Recipient (relevant genotype)

Donor (relevant type)

geno-

Selection

Transductants Sample Class

hisH2 tyrAl trpE26 Tre+

tre-12 trpE26

175

Cotrans(%) duction

No.

Tre+ His+ Tre+ HisTre+ Tyr+ Tre+ Tyr-

98 77 98 77

44.0

44.0

tre-12 trpE26

metB3 trpE26

Tre+

312

Tre+ Met+ Tre+ Met-

277 35

11.2

hisH2 trpE26

tre-12 trpE26

His+

603

His+ TreHis+ Tre+

354 249

58.7

metB3 trpE26

tre-12 trpE26

Met+

229

Met+ Tre+ Met+ Tre-

199 30

13.1

ilvA2 trpE26

nov-3 trpE26

Ile+

203

Ile+ NovR Ile+ Novs

135 68

665

metB3 trpE26

nov-3 trpE26

Met+

203

Met+ NovR Met+ Novs

126

Hom+ Ile+ Hom+ Ile-

212139

hom-1 trpE26

ilvA2 trpE26

Hom+

351

2.1

62.

39.6

"For other mapping data on this region see (12).

tre-12

homla hisH2 tyrAl metB3 ilvA2 nov-3 thr-5 aid

8 r ~~~~~~86.7 i i | 86.7 | j ~~~~71.9 j

|

- 62.8-

4-3j-937 9

.:

~

j

I88.8------I I

~~~ ~~I

I

,

FIG. 1. Map of the tre-12-ald region of the chromosome in trpE26 strains. The map is constructed from the results oftwo-factor PBS1 -mediated transduction crosses of Table 2, and from those of an earlier publication (12). The arrows point to the selected markers. Figures are of (1-cotransduction frequency) x 100. (a) thr-5 and hom-1 are depicted as a single marker as hom-1 is a deletion extending into the thr region. (b) In this cross the selected marker was hom-1+. thr-5 was used in all other crosses involving the hom-1-thr-5 site.

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TABLE 3. Linkage relationships of markers in the cysB-argC region of the chromosome of trpE26 strains measured in PBS1 transduction experimentsa Recipient (relevant genotype)

Donor (relevant genotype)

Selection

Transductants Class Sample

No.

Cotransduction (%)

cysB3 trpE26

catA trpE26

Cys+

318

Cys+ Prt+ Cys+ Prth

290 28

8.8

metDl trpE26

catA trpE26

Met+

118

Met+ Prt+ Met+ Pfth

75 43

36.4

cysB3 trpE26

gly-133 trpE26

Cys+

198

Cys+ Gly+ Cys' Gly-

181

8.6

Cys+ Gly-

154

Cys+ Gly+

18

cysB3 gly-133 trpE26 trpE26

gly-133 metDl trpE26 cysB3 trpE26

gly-133 metDl trpE26 argC4 trpE26

Gly+

Met+

172 162

393

cysB3 metDl trpE26 gly-133 trpE26

Met+

114

argC4 trpE26

gly-133 trpE26

Arg+

73

cysB3 trpE26

thi-86 trpE26

Cys+

102

thi-86 trpE26

Gly+

obtained from crosses involving the thi-86 marker. The map of the cysB3-argC4 region in trpE26 strains (Fig. 2) is based on the above results. Taken together the results of experiments so far carried out on mapping in trpE26 strains mean that these strains differ from strain 168 by the translocation of the trpD-ilvA segment to a position next to thr as well as by the inversion of the whole top section of the chromosome. Tre-12 is now linked to the left hand end point of the translocation while cysB3 is joined to the gly region. The complete maps of the two types of strains are presented in Fig. 3. We have used a system of letters and numbers to label sections of the chromosome. Thus the map of strain 168 has A to D on its left-hand side, and I and II on the right-hand side. In trpE26 strains section C (trpD-ilvA) is shifted to the origin-proximal end of section B, and the segment A-I is inverted, so that A now lies next

106

10.5

Gly+ Gly+ Gly+ Gly+

Cys+

141

CysMetMet+

21

13.0

95 67

41.4

Met+ Met+ Met' Met+

GlyGly+ ArgArg+

222 171 333 60

Met+ Gly+ Met+ Gly-

68 46

40.4

Arg+ Gly+ Arg+ Gly-

64 9

12.3

Cys+ ThiCys+ Thi+

57 45

55.9

83 Gly+ Thi+ 23 Gly+ ThiPrth, phenotype of catA clones; overproduction of protease (see Materials and Methods).

gly-133 trpE26 a

Cys+

17

43

84.7

21.7

to II, and I next to C. Since the inverted segment contains the origin of replication and C is translocated in its normal orientation, chromosome replication in all regions is in the usual direction. To ascertain whether all the haploid trpE26 strains at our disposal had the same chromosome structure we constructed a trpE26 strain bearing the hisH2 and tre-12 markers. This strain was used as a recipient in coupling transduction crosses with three different trpE26 strains as donors: 166, GSY468, and GSY1269. The latter two strains were constructed in two separate experiments by the same transformation cross: recipient, aroB2 ilvC1; donor, trpE26 (strain 166). HisH2 and tre-12 were found to be linked in this way with all three donors, but not with a wild-type donor (of strain 168 genetic background). These results are recorded in Table 4. Unstable merodiploids from strains of 168


VOL. 126, 1976

cysB3 thi-86 [ly-133 catAl

613

metDl argC4 a

, >44.1-: 78.3 { 87. 0 90.4 912 | I

H-63.6-----63.6~~~~58.0 58.6

10

1

-

I

i ,_~~- -

.15.387 7

FIG. 2. Map of the cys-B3-argC4 region in trpE26 strains. Gly-133 and catA are shown in brackets since the relative order of these loci is not yet known. The map is constructed from results of the two-factor PBS1mediated transduction crosses shown in Table 3. The arrows point to the selected markers. Figures are of (1cotransduction frequency) x 100.

I

A

168

1:0

A

166 (and other trp.E 26strains)

FIG. 3. Genetic maps of the chromosomes of strain 168 and strains carrying the trpE26 mutation. For clarity only a few relevant markers are shown. 0 and T: origin and terminus of replication. The thickened region represents the translocated segment and the open squares mark the positions in which strains differ in linkage relationships. A-D and I-II indicate linkage groups of markers (respectively of the left- and righthand halves of the 168 strain chromosome) which are conserved in both types of strains. (For further details see text.)

genetic structure. The existence of a translocation in trpE26 strains has led to the elaboration of a model for the formation of unstable merodiploids when such strains are transformed or transduced by strains of 168 genetic structure (12). Formation of merodiploids by this mechanism is not, however, necessarily restricted to strains bearing the particular trpE26 mutation. The model requires only that the two partner strains differ in the relative order of two

segments on the chromosome, and that selection is made for markers at the junction of these segments in the donor. This results in the joining of two genetic elements of the recipient by the exogenous DNA fragment, to give a chromosome possessing a tandem duplication. The model therefore predicts that one should be able to obtain merodiploids from 168 type recipients with trpE26 strains as donors. This rationale is illustrated in Fig. 4. The recipient carries two

614

J. BACTERIOL.


the recipient they are located near the end points of the region covered by the two segments the order of which is different in the two partners. The model of Fig. 4 is therefore the same as that previously proposed for the trpE36

markers, ilvAl and hom-i. The double Ile+ Hom+ transductants should bear a long tandem duplication for the region hom-i to ilvA since the two markers are closely linked in the donor but widely separated in the recipient. In fact, in

TABLE 4. Linkage of tre-12 and hisH2 in coupling PBS1 transduction crosses with a trpE26 recipient and three different trpE26 donors Transductants

Recipient (relevant gen- Donor (no. and relevant genotype) otype)

a

Selection

Sample

Class

No.

Cotransduction (%)

tre-12 hisH2 trpE26

166 trpE26

His+

120

His+ Tre+ His+ Tre-

52 68

43.4

tre-12 hisH2 trpE26

GSY468 trpE26

His+

120

His+ Tre+ His+ Tre-

62 58

51.7

tre-12 hisH2 trpE26

GSY1269 trpE26

His+

120

His+ Tre+ His+ Tre-

62 58

51.7

tre-12 hisH2 trpE26

WTIl

His+

80

His+ Tre+ His+ Tre-

0 80

0.0

WT, Wild type.

hisA cysB hom I

I

a

aroB citK trpC ilvA Ii I I

leuI

4-

T

DNA(trp E26 ) I

I

hisA

cysB hom

Ieu

I

I

trpC IsA citK aro B

hisA cysB hom I1 I

I

aroB trpCa llvA+homr I I.

leua

1hom+

.1

leu a

.

aro B citK .trpC ivA I I

a

aroB trpC ilvA citK

leu

/

I/

21

31

trpC ilvA' aroB FIG. 4. Models for the formation and segregation ofmerodiploids in crosses involving strains of168 genetic structure as recipients and trpE26 strains as donors. The example shown is a PBS1 transduction cross: donor trpE26; recipient hom-i ilvAl. Only a few relevant markers are shown on the left half of the recipient chromosome. The two genetic elements joined could be either two independent chromosomes or two arms of a single chromosome in the course of replication. The lower part of the diagram shows the postulated mechanism for the formation of segregants. There is pairing of the two homologous regions and single recombination events at: 1, to the left of hom; 2, between hom and ilvA; 3, to the right of ilvA. The outcome of such events is discussed in the Results.

hisA

cysB hom

leu


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TABLE 5. Cotransduction of hom-1 and ilvAl with a 168 M recipient and a trpE26 strain as donor Recipient (deriva- Donor (derivation and releTransductants Cotranstion and relevant duction vat andtre) Selection genotype) Sample Class No. (%)

(168M)a hom-1 ilvAl

(GSY468) hom+ ilvA+ trpE26

Ile+

156

Hom+

297

Ile+ HomIle+ Hom+ Hom+ IleHom+ Ile+

90 66 231 66

42.3

Ile+ HomIle+ Hom+ Hom+ IleHom+ Ile+

83 0 68 0

0.0

(GSY468P (GSY468) Hom+ 351 Hom+ Ile+ hom-1 trpE26 ilvA2 trpE26 Hom+ Ilea In this cross the Hom+ Ile+ transductants are merodiploid (see text). ^ Control crosses.

212 139

(168M)P hom-1 ilvAl

(168M) trpC2

Ile+

83

Hom+

68

(recipient) merodiploids, although in that case selection for a single marker, Trp+, is sufficient (12). The results of the first experiment of this kind are shown on Table 5. They show that hom-1 and ilvAl can be cotransduced when in a 168 recipient if the donor is a trpE26 strain. Moreover the linkage values for this cross are remarkably close to those obtained with a trpE26 recipient; about 40% (experiment 3, Table 5). It should be noted that the two markers are not directly linked in strain 168 (experiment 2, Table 5). According to the model of Fig. 4 the Ile+ Hom+ transductants in such crosses should be unstable merodiploids, redundant for the region extending from hom to ilvA in strain 168. We have not made a thorough analysis of the extent ofthe diploidized region in these strains. However, the fact that they segregated auxotrophic clones, and their segregation pattern, indicated that they were merodiploids of the predicted structure (Fig. 4). Ile+ Hom+ transductants were purified by two successive clonings on selective medium, single colonies were resuspended in MG, diluted, and spread onto MG plates supplemented with isoleucine, threonine, and methionine. These plates were then replicated to determine the phenotypes of the colonies. Twenty-eight out of 30 transductants so tested segregated to give clones ofall the four phenotypes shown on Table 6 (results for five of these transductants are given on the table). The relative proportions of each class of segregants are of significance. The two classes of low frequency (Hom+ Ile- and Hom+ Ile+) must have arisen by crossovers respectively at positions 1 or 3 (Fig. 4) where there is only a short length of homology. The much larger class (Hom- Ile-) is obtained from a crossover at

22.2

0.0 39.6

TABLE 6. Segregation of Hom+ Ile+ clones from a transduction cross of a 168 (hom-1 ilvAl) recipient and a trpE26 donore Phenotypes of colonies tested No. of clone

No. of colonies tested

1 2 3 4 5 Total Percent of total

130 116 240 110 71 667 100

a

Hom+ Ile+

89 69 162 77 52 449 67.3

Hom- Hom+ Ile+ Ile-

2 2 4 5 2 15 2.3

1 6 5 2 6 20 3.0

HomIle-

38 39 69 26 11 183 27.4

Transductants from the first cross of Table 5.

position 2 (i.e., at any position between hom and ilvA). The remaining class, Hom+ Ile+, is of high frequency, as expected, since it is mostly composed of clones still remaining diploid. This was demonstrated by examining their continued segregation. Four clones from each of the first three classes of segregants of Table 6 were tested for this property, in the same manner as above. Only the Hom+ Ile+ clones produced segregants. All the Hom+ Ile+ transductant clones were found to be inhibited by lysine when they were streaked onto MG plates containing this amino acid. Lyssen is a phenotype of unstable, and certain stable trpE26 merodiploids (2, 3). It is thought to be due to a redundancy of a part of section B of the chromosome. This point will be discussed later. The Hom+ Ile- transductant clones were all resistant to lysine. The HomIle+ clones could not be tested as the inhibitory effect of lysine is overcome by the growth requirements of these strains (threonine and methionine). Some further crosses showed that merodip-

616


loids eould be obtained from strain 168 type recipients even when selection was for only one marker. In Table 7 are the results of two experiments in which Thr+ transductants were selected, in the presence or absence of lysine, using trpE26 donor lysates. About 50% of the transductants were Lyssen on selection. However this property was rapidly lost. When retested by streaking onto MG plates 47 Thr+ transductants (taken from the plates without lysine; Table 7, second cross) were all phenotypically Lysees, Ile+. Thirteen of these clones were then purified once and spread to give single colonies on MG plates containing threonine and isoleucine. Samples of at least 100 colonies from each clone were replicated to test their phenotypes. In no case were there any Lyssen or Ilecolonies, These results indicate that these initial merodiploids segregated rapidly after selection for threonine independence to give haploid Thr+ Ile+ recombinants. The third cross of Table 7 shows that Lyssen transductants were also obtained when selection was only for Ile+. In this case 77.4% of the transductants were sensitive to lysine. On purification these clones also became stable and Lyse. These results confirm the view that in crosses of this type segregation by the mechanism shown in Fig. 4 must be rapid. DISCUSSION The studies on the replication order of markers and the transduction mapping data reported previously (12), as well as in the present paper, are all compatible with the structure of the chromosome of trpE26 strains shown in Fig. 3. The chromosome of these strains differs therefore from that of strain 168 by a translocation and an inversion. Following the designations for the chromosome sections adopted in Fig. 3 the two halves of the strain 168 chromoTABLE 7. Sensitivity to L-lysine among Thr+ or Ile+ transductants from crosses involving 168 recipients and trpE26 strains as donors Recipient

Transductants

Door(dt vat ion and

Percent No. + Selec- No. on MG onLyw tion MG

(168) thr-5

(GSY468) trpE26

Thr+

692

324

53.2

(168) thr-5

(GSY468) ilvA2 trpE26

Thr+'

296

152

48.7

(deriva-

nd relevant gentionvand reeat otype)

genotype)

LyM~ iL-lysine

(168) Ile+ 399 90 77.4 (GSY468) ilvAl trpE26 " In this cross isoleucine was also included in the selection medium.

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J. BACTERIOL.

some are A-B-C-D and I-II, whereas the two halves of a trpE26 strain chromosome are I-CB-D and A-H. Therefore, to pass from the strain 168 chromosome to that of a trpE26 strain four breaks (and rejoinings) are necessary. The positions of the breaks are now fairly accurately known: (i) inside the trpE26 locus; (ii) between cysB and nov (iii); between ilvA and citK; (iv) between tre and thi. There is evidence that all three trpE26 strains that we have tested have the same structure. These are the original trpE26 mutant (strain 166), strain GSY468, and strain GSY1269. The last two strains were obtained by introduction of the trpE26 marker of strain 166 into recipients of 168 origin. The results from which this conclusion is drawn are the following. (i) Used as donors in transduction crosses all three strains revealed the linkage of tre-12 to hisH2 in trpE26 recipients (Table 4). This shows that in all of them section I is directly attached to section C. (ii) The same three donors failed to show the cotransduction of cysB3 and thr-5 in a recipient of 168 genetic structure. Thus none of them has the A-B junction. (iii) The replication order of markers was the same in two trpE26 strains analyzed by use of the density transfer technique (strains 166 and GSY468). One wonders how the chromosomal abberations that determine the trpE26 marker are brought about. For the original 166 strain where spores were irradiated by X-rays, this seems understandable (4). However, as we have shown, the trpE26 marker has been repeatedly introduced by transformation of the aroB2 marker of strain 168 (resulting in strains GSY468 and 1269). The mechanism of this transmission needs explaining. We previously considered a tentative model (12). This involved excision of segment C in the forn of a circle and consecutive integration of the circle next to the thr region. It explained the creation of junctions C to B and B to D in the trpE26 strains (Fig. 3). We had postulated the existence of regions of partial homology in distant areas of the B. subtilis chromosome which would aid these events. In view of the discovery of the inversion of the A-I segment the model should now be modified. The possibility of further homologies has to be considered at the junctions III and A-B of the 168 chromosome. Although these points are still unclear the outcome (the creation of strains GSY468 and 1269) suggests that dramatic exchanges of segments of chromosome do take place in these regions. That these exchanges occur at determined areas is indicative of pairing of similar deoxyribonucleic acid (DNA) sequences. It is tempting to

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speculate which factors may be the cause of the regions of partial or complete homology postulated. The presence of several copies of a defective phage, insertion sequences (such as have been demonstrated in Escherichia coli [11]), sites for restriction endonucleases, or even longer palindromes (10) could be mentioned as possibilities. The view that regions of homology are implicated in the exchanges of chromosome segments (translocation and inversion) considered above was strengthened by recent data on the genetic structure of a certain class of stable merodiploids arising from unstable merodiploids of trpE26 strains. In these stable clones loss of the copy of segment C nearest to the origin resulted also in the return of the A-I segment to its orientation in 168 strains: section A is reunited to B and section I to II (Trowsdale and Anagnostopoulos, unpublished data). Apparently then the A-I segment is able to switch back and forth. Such a phenomenon could be accounted for by the bringing together of homologous regions. A minor observation made in the course of the work reported in the present paper may be of relevance. This concerns the construction of strain GSY2205 (trpE26 thi-86) through transduction of GSY1992 (trpE26 tre-12) by a lysate of a 168 type strain. At that time the position of the thi-86 marker in trpE26 strains was not known. This marker was introduced among the Tre+ transductants at low frequency (1 to 2%). It is now known that thi-86 is not directly linked to tre-12 (section I) but to cysB3 (section A, Table 3; Fig. 2) in these strains and must lie near one end point of segment II since it is linked to gly-133 in both types of strains. In the clones that have acquired thi-86 the incoming DNA piece may have paired simultaneously with the tre region of segment I and the thi region of segment II, situated in different halves of the chromosome. Through a quadruple recombination event these clones acquired tre-12+ and thi-86. Altematively the DNA piece carrying these two markers may have been fragmented after entering the cell, and the two markers integrated independently. The demonstration that merodiploids are readily obtained in crosses with 168 type recipients and trpE26 strains as donors, when selection is done for markers near the C-B junction, strongly supports the proposed model for the formation of merodiploids (12). As can be seen from Fig. 4, redundancy is of the same length and always concerns the same two segments, B and C. The diploids bear a tandem duplication of about one-third of the chromosome. The order of the two copies of the duplication is of

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course different in the two kinds of diploids: those from trpE26 strains C-B-C-B and those from 168 strains B-C-B-C. The generality ofthe model for strains differing by a translocation is evident. It must also be recalled that the same model has been considered for the transmission of tandem duplications and, with a slight modification, for the generation of a tandem duplication when the recipient carries a deletion (6, 8). The remarkably high frequency of appearance of merodiploids from 168 recipients in the crosses of Tables 5 and 7 needs special mention. It should be noted that the trpE26+ transformants, all of which are merodiploid, also appear with high frequency (2). This adds weight to the suggestion, made in the preceding paper, that if the model is correct fusion of two genetic elements occurs quite often in B. subtilis transformation and transduction (12). The fact that the merodiploids from 168 strains are, like those made from trpE26 strains, Lyssen, brings some additional infornation concerning the lysine effect. The common element in all lysine-sensitive strains is redundancy of segment B. Stable merodiploids in which only the C segment is redundant are Lysres (1, 3). Recent data indicate that lysine sensitivity is determined by the diploid state of a locus, not yet identified, close to the left-hand end point of the B segment. ACKNOWLEDGMENT J. Trowsdale was a Unilever European Fellow of the Biochemical Society.

LITERATURE CITED 1. Audit, C., and C. Anagnostopoulos. 1973. Genetic studies relating to the production of transformed clones diploid in the tryptophan region of the Bacillus subtilis genome. J. Bacteriol. 114:18-27. 2. Audit, C., and C. Anagnostopoulos. 1972. Production of stable and persistent unstable heterogenotes in a mutant ofBacillus subtilis, p. 117-125. In H. 0. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores V. American Society for Microbiology, Washington, D.C. 3. Audit, C., and C. Anagnostopoulos. 1975. Studies on the size of the diploid region in Bacillus subtilis merozygotes from strains carrying the trpE26 mutation. Mol. Gen. Genet. 137:337-351. 4. Burkholder, P. R., and N. H. Giles. 1947. Induced biochemical mutations in Bacillus subtilis. Am. J. Bot. 34:345-348. 5. Harford, N. 1975. Bidirectional chromosome replication in BaciUus subtilis 168. J. Bacteriol. 121:835-847. 6. Hill, C. W., D. Schiffer, and P. Berg. 1969. Transduction of merodiploidy: induced duplication of recipient genes. J. Bacteriol. 99:274-278. 7. Jamet, C., and C. Anagnostopoulos. 1969. Etude d'une mutation trbs faiblement transformable au locus de la thr6onine d6saminase de Bacillus subtilis. Mol. Gen. Genet. 105:225-242. 8. Jamet-Vierny, C., and C. Anagnostopoulos. 1975. Induction and transmission of a merodiploid condition near the terminal area of the chromosome of BaciUus

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subtilis. Genetics 81:437-457. 9. Lepesant-Kejzlarova, J., J. A. Lepesant, J. Walle, A. Billault, and R. Dedonder. 1975. Revision of the linkage map of Bacillus subtilis 168: indication for circularity ofthe chromosome. J. Bacteriol. 121:823-834. 10. Sobell, H. 1973. Symmetry in protein-nucleic acid interaction and its genetic implications. Adv. Genet. 17:411-490. 11. Starlinger, P. and H. Saedler. 1972. Insertion mutations in micro-organisms. Biochimie 54:177-185. 12. Trowsdale, J., and C. Anagnostopoulos. 1975. Evidence

J. BACTERIOL. for the translocation of a chromosome segment in Bacillus subtilis strains carrying the trpE26 mutation. J. Bacteriol. 122:886-898. 13. Trowsdale, J., and D. A. Smith. 1975. Isolation, characterization and mapping of Bacillus subtilis 168 germination mutants. J. Bacteriol. 123:83-95. 14. Young, F. E., and G. A. Wilson. 1975. Chromosomal map of Bacillus subtilis. p. 596-614. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI, American Society for Microbiology, Washington, D.C.