APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1977, Copyright 1977 American Society for Microbiology

p.

989-993

Vol. 33, No. 4 Printed in U.S.A.

Construction of a Kit of Reference Strains for Rapid Genetic Mapping in Bacillus subtilis 168 R. A. DEDONDER,* J-A. LEPESANT, J. LEPESANT-KEJZLAROVA, A. BILLAULT, M. STEINMETZ, AND F. KUNST Unite de Biochimie Cellulaire, Institut de Recherche en Biologie Moleculaire, Centre National de la Recherche Scientifique and Universite Paris VII, 75221 Paris Cedex 05, France

Received for publication 9 December 1976

A set of nine reference strains bringing convenient markers in the genetic background of Bacillus subtilis Marburg 168 has been prepared to allow rapid mapping of new markers. A reexamination of the genetic linkage data in Bacillus subtilis (1, 10) based on bidirectional deoxyribonucleic acid replication (3, 7, 9) led our group to the formulation of a continuous

Marker a Auxotrophic aroD120 aroG932 aroI906 ctrAl

cysA14 dall gltA292 glyB133 hisAl ilvAl leuA8 lysi metC3 purA16 purB33 pyrDl thr5 thyAl thyBl d trpC2 Other aldl

circular chromosomal map, which is currently accepted (4, 6, 8). At the first meeting on Bacillus organized by the American Society for Microbiology (Ithaca, N.YJ, 6-9 August 1975),

TABLE 1. Description of the genetic markers of the kit strains Selection of recombinants Requirementb Phenylalanine + tyrosine + tryptophan Phenylalanine + tyrosine Phenylalanine + tyrosine + tryptophan Cytidine in the absence of ammonium Cysteine i-Alanine Glutamate (or aspartate) Glycine Histidine Isoleucine Leucine Lysine Methionine Adenine Adenine (or guanine or hypoxanthine) Uracil Threonine Thymine Tryptophan

MMG mediumc MMG medium MMG medium MMG medium in which ammonium is replaced by 0.1% glutamate MMG medium TBAB medium MMG medium MMG medium MMG medium MMG medium MMG medium MMG medium MMG medium MMG medium MMG medium MMG medium MMG medium MMG medium MMG medium

MM medium + 0.1% L-alanine + 0.0015% Casamino Acids C medium + 0.1% sucrose Sucrosee sacA321 MM medium + 0.1% trehalose + 0.0015% Trehalosee trel2 Casamino Acids a For origin, characterization, and localization of markers, see references 8 and 11. b Added to MMG medium to a final concentration of 20 gg/ml, except for thymine (50 ,ug/ml) and glutamate or aspartate (400 Ag/ml). For growth of strains carrying the ctrAl, dali, or thyAl thyBl markers, even rich media such as TBAB need to be supplemented with cytidine, D-alanine, or thymine, respectively. c Media: MM, Spizizen mineral medium; MMG, MM + glucose, 0.1%; C, mineral medium with a lower citrate concentration (0.07 mM; 7); TBAB, standard tryptone blood agar base. d Single mutants thyAl or thyBl are phenotypically Thy+. e Inability to use this component as a carbon source. 989 L-Alaninee

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NOTES

where we presented the reassessment of some of the weak linkages previously described (N. Harford, J. Lepesant-Kejzlarova, J-A. Lepesant, R. Hamers, and R. Dedonder, Microbiology-1976, p. 28-34, American Society for Mi-

crobiology, Washington, D.C., 1976), asked to prepare

FIG. 1. Simplified genetic map of B. subtilis 168 and chromosomal segments defined by the limits between which a given unlocated marker can be mapped in transduction crosses with kit strains no. 1 to 9 as donors or recipients.

we were

set of reference strains allowing rapid mapping of new markers in B. subtilis 168. Some of the difficulties encountered in the construction of the chromosomal map of B. subtilis originated obviously in the use of heterogenic strains, such as strains W23 and 168. This communication describes the construction of a set of isogenic strains that we hope will be suitable for mapping of new mutations in B. subtilis 168. A number of well-mapped and widely distributed and used markers have been chosen as reference markers. The basic principles that guided the choice were: (i) reduce the number of markers per strain to a minimum in order to reduce the side effects on competence, motility, sporulation, etc., still allowing a sufficient overlap in transduction experiments with a

TABLE 2. Construction of strain QB123 (kit no. 9) Strain

Genotype

Origin Trp+ transformant of strain 168 ctrAl trpC2 S. A. Zahler and P. Winter thr5 leuA8 metB5 N. Sueoka SB3 cysB3 hisAl trpC2 E. Nester SB25 hisB2 trpC2 E. Nester SB26 metC3 trpC2 E. Nester QBla sacA321 trpC2 Reference 7 QB97 hisB2 BM2 i SB25 QB99 hisB2 leuA8 Mu8u5u5 + QB97b i168 QB101 metC3 leuA8 trpC2 SB26 A QB99 QB102 metC3 hisAl trpC2 SB3 4 QB101 QB103 sacA321 hisAl trpC2 QB1 QB102 QB123 sacA321 ctrAl trpC2 CU479 4 QB103 a Strains were constructed in this laboratory by congression of markers at saturating concentrations of deoxyribonucleic acid (4) or by PBS1 transduction (n). Arrows point from the donor to the recipient. bA mixture of deoxyribonucleic acids extracted from strains Mu8u5u5 and QB97 was used in this cross.

BM2 CU479 Mu8u5u5

TABLE 3. Construction of strain QB917 (kit no. 8)

TABLE 5. Construction of strain QB928 (kit no. 2)

Strain Genotype Origina QB666 sacA321 hisAl leuA8 thr5 Mu8u5u5 it QB103 QB917 hisAl thr5 trpC2 168 i QB666 a See footnote a, Table 2, for explanation of tf.

Strain Genotype Origina Mu33 purB33 N. Sueoka WB906b aroI906 J. A. Hoch QB811 sacA321 keuA8 metB5 dall Reference 8 tf QB811 QB822 sacA321 leuA8purB33 dali Mu33 tf QB925 leuA8 purB33 dali trpC2 168 t QB822 tf QB925 QB928 aroI906 purB33 dali trpC2 WB906 -f a See footnote a, Table 2, for explanation of tf. ° This strain also carries a strr marker.

TABLE 4. Construction of strain QB922 (kit no. 5) Strain

Genotype

Origin

GSY292 gltA292 trpC2 J. A. Hoch QB922 gltA292 trpC2 GSY292 it QB103a a See footnote a, Table 2, for explanation of tf.

VOL. 33, 1977

NOTES

991

TABLE 6. Construction of strain QB934 (kit no. 3) Strain

Proz. 1 GSY289 QB163 QB668 QB687 QB693 QB809 QB810 QB827 QB835

QB917b

a

QB923 QB932 QB934 See footnote a,

Genotype

TABLE 7. Construction of strain QB935 (kit no. 6) Strain GSY254 SB120 QB15 QB122 QB929 QB935 a

Origina

recH342 thi78 glyB133 met29 his2 pyrDI trpC2 metC3 pyrDl trpC2 sacA321 hisAl leuA8 metB5 sacA321 trel2 trpC2 purB6 trel2 purB33 trpC2 purB33 sacA321 leuA8 metB5 purB33 sacA321 leuA8 trel2 purB33 glyB133 trel2 sacA321 sacA321 hisAl thr5 trpC2 sacA321 metC3 hisAl trpC2 trel2 metC3 hisAl trpC2 trel2 metC3 glyB133 trpC2 Table 2, for explanation of tf.

Genotype lysl trpC2 aroD120 trpC2 sacA78 sacA78 lysl aroD120 hisAl trpC2 aroD120 lysl trpC2

Origina C. Anagnostopoulos N. Harford Reference 7 QB15 tf GSY254 SB120 t QB103 QB122 g QB929

See footnote a, Table 2, for explanation of tf.

phage PBS1; and (ii) retain easily selectable markers, with preference given to auxotrophic markers so that most of the mapping can be done with Spizizen mineral medium. We chose the following markers and distributed them into nine groups: purA16 cysA14; aroI906 purB33 dall; trel2 metC3 glyB133; pyrDl ilvAl thyAl thyB1; gltA292; aroD120 lysl 1euA8; aroG932 aldl; hisAl thr5; sacA321 ctrAl. These markers are designated according to the last compilation on B. subtilis genetics of Young and Wilson (11), where their origin and former nomenclature can be found. A short description is presented in Table 1. The positions of these markers on the map are shown in Fig. 1.

The following remark must be made about the cysA14 marker. cysA14 was extracted from strain M23. In this strain the cysA locus behaves as a complex locus containing several mutations (5). From strain M23, we obtained strain N23 as a methionine-independent transformant able to grow on minimal medium supplemented with adenine, cysteine, and isoleu-

A. Prozorov C. Anagnostopoulos GSY289 tf tf QB101 Reference 8 Reference 8 Reference 8 Mu33 tf QB103 t- QB668 QB809 tf tf QB810 QB687 tf tf QB827 Proz. 1 tf 168 tf-* QB666

QB163 If> QB917b QB693 tf-f QB923 QB835

tf

QB932

cine. The cysA marker from this strain has been introduced into strain QB944. From the results of phenotypic tests and backcrosses with this strain, we are tempted to conclude that it is free from the original additional mutations in the cysA locus (Cyss, Cym-, or H2S+). The chosen markers have been introduced into the genetic background of the 168 strain by congression of markers at the saturating concentration of deoxyribonucleic acid or by transduction with phage PBS1. Tables 2 through 10 show the construction of the nine strains of the kit, and Table 11 summarizes their nomenclature and genotypic characteristics. None of the markers present in the strains kit 1 to 9 produces side effects on sporulation, transformability, or transducibility: the strains can be maintained as spores on AK slants (BBL) (it is often advisable to supplement AK medium with auxotrophic requirements to obtain efficient sporulation); the strains are normally transformable under our routine conditions (frequency range, 0.1 to 2%; in the case of strain kit no. 4 transformation conditions were modified according to Farmer and Rothman [2]); and the strains are normally transducible. In two- or three-factor transduction crosses, using these strains as recipients, the order of the markers on the map and the distances expressed as a percentage of recombination were always in agreement with those obtained earlier by Lepesant-Kejzlarova et al. (8) and by Harford et al. (Microbiology-1976, p. 28-34). Some of the results obtained in such crosses are presented in Tables 12 and 13.

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APPL. ENVIRON. MICROBIOL.

NOTES TABLE 8. Construction of strain QB936 (kit no. 7)

Origina

Genotype

Strain

aroG932 WB932b sacQ36 aldl trpC2 QB698 leuA8 metB5 sacA321 argA2 QB818 leuA8 argA2 sacA321 trpC2 QB920 leuA8 aroG932 sacA321 trpC2 QB931 leuA8 aroG932 aldl trpC2 QB936 a See footnote a, Table 2, for explanation of tf and td. b This strain also carries a strr marker.

J. A. Hoch Reference 8 Reference 8 168- QB818 WB932 QB920 QB698 tf QB931

TABLE 9. Construction of strain QB943 (kit no. 4) Genotype

Origina Strain F. Rothman 168TT thyAl thyBl trpC2 Reference 8 sacA78 ilvAl QB16 sacA321 hisAl ilvAl trpC2 QB16 .t.fM QB917b QB921 QB163 tf, QB921 pyrDl hisAl ilvAl trpC2 QB926 168TT tf, QB926b pyrDl hisAl ilvAl thyAl thyBl trpC2 QB933 168 -t QB933 pyrDl ilvAl. thyAl thyBl trpC2 QB943 a See footnote a, Table 2, for explanation of tf. b Selection for methotrexate resistance and screening for thymine requirement.

TABLE 10. Construction of strain Strain Genotype purA16 cysA14 metB5 ilvAl M23 purA16 leuA8 metB5 Mu8u5u16 purA16 hisAl trpC2 QB197 sacA321 QB1072 N23 purA16 cysA14 ilvAl QB944 purA16 cysA14 trpC2 a See footnote a, Table 2, for explanation of tf.

QB944 (kit no. 1) Origina D. Karamata N. Sueoka Mu8u5ul6 tf QB102 Derives from QB103 by transformation QB1072-f M23 N23 tf-+QB197

TABLE 11. Nomenclature and characteristics of the reference strains Strain

Kit no. 1 2 3

Collection no. QB944 QB928 QB934

4

QB943

5 6 7 8

QB922 QB935 QB936 QB917 QB123

9

Genotype

purA16 cysA14 trpC2 aroI906 purB33 dall trpC2 trel2 metC3 glyB133 trpC2 pyrDl ilvAl thyAl thyBl trpC2 gltA292 trpC2 aroD120 lysl trpC2 leuA8 aroG932 aldl trpC2 hisAl thr5 trpC2 sacA321 ctrAl trpC2

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VOL. 33, 1977

TABLE 12. Transduction cross with strain kit no. 7 as recipient Donor

Recipient

QB300, sacQ36 a

Kit no. 7, leuA8 aroG932

trpC2

a

aldl trpC2

Linked markers

tion

AId+

aldl-sacQ36

aroG932-leuA8

Aro+

aroG932-sacQ36

Aro+

leuA8-aroG932

Leu+

Recombi-

Cotransfer Ald+ Lvsh Ald+

180

239

25

31 AroLeu Aro+ 239 4 Aro+ LVSh = Aro 40 Leu+ Aro+ 2e8 Leu+ 23

87

98 83

83

The sacQ36 allele leads to overproduction of levansucrase (Lvsh phenotype).

TABLE 13. Transduction cross with strain kit no. 9 as recipient Donor

QB54, sacS49a purA16

Recipient

Linked markers

Kit no. 9, sacA321 ctrAl trpC2

tAlA321

nation (%)

Cotransfer

Ctr+ Suc+ Ctr+

119

240

50

Ctr=Co 40 73 240 Ctr+ 6 Suc Con 33 Suc+ sacA321-sacS49 239 Suca T The sacS49 allele leads to constitutive synthesis of sucrase and levansucrase (Con phenotype).

ctrAl-sacS49

a

Selection

LITERATURE CITED 1. Dubnau, D. 1970. Linkage map of Bacillus subtilis, p. 39-45. In H. A. Sober and R. A. Harte (ed.), Handbook of biochemistry, 2nd ed. Chemical Rubber Co. Press, Cleveland, Ohio. 2. Farmer, J. L., and F. Rothman. 1965. Transformable thymine-requiring mutant of Bacillus subtilis. J. Bacteriol. 89:262-263. 3. Gyurasits, F. B., and R. G. Wake. 1973. Bidirectional chromosome replication in Bacillus subtilis. J. Mol. Biol. 73:55-63. 4. Harford, N. 1975. Bidirectional chromosome replication in Bacillus subtilis 168. J. Bacteriol. 121:835-847. 5. Kane, J. F., R. L. Goode, and J. Wainscott. 1975. Multiple mutations in cysA14 mutants of Bacillus subtilis. J. Bacteriol. 121:204-211. 6. Kejzlarova-Lepesant, J., N. Harford, J-A. Lepesant, and R. Dedonder. 1975. Revised genetic map for Bacillus subtilis 168, p. 592-595. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI. American

Ctr+

Society for Microbiology, Washington, D.C. 7. Lepesant, J-A., F. Kunst, J. Lepesant-Kejzlarova, and R. Dedonder. 1972. Chromosomal location of mutations affecting sucrose metabolism in Bacillus subtilis

Marburg. Mol. Gen. Genet. 118:135-160. 8. Lepesant-Kejzlarova, J., J-A. Lepesant, J. Walle, A. Billault, and R. Dedonder. 1975. Revision of the linkage map ofBacillus subtilis 168: indications for circularity of the chromosome. J. Bacteriol. 121:823-834. 9. Wake, R. G. 1973. Circularity of the Bacillus subtilis chromosome and further studies on its bidirectional replication. J.Mol. Biol. 77:569-575. 10. Young, F. E., and G. A. Wilson. 1974. Bacillus subtilis, p. 69-114. In R. C. King (ed.), Handbook of genetics, vol. 1. Plenum Press, New York. 11. Young, F. E., and G. A. Wilson. 1976. Revision of the linkage map of Bacillus subtilis, p. 686-703. In G. D. Fasman (ed.), Handbook of biochemistry and molecular biology, 3rd ed., vol. II. Chemical Rubber Co. Press, Cleveland, Ohio.

Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168.

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1977, Copyright 1977 American Society for Microbiology p. 989-993 Vol. 33, No. 4 Printed in U.S.A. Co...
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