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Vol. 172, No. 2JOURNAL OF BACTERIOLOGY, Feb. 1990, p. 793-8010021-9193/90/020793-09$02.00/0Copyright © 1990, American Society for Microbiology
Genetic Map of the Bacillus stearothermophilusNUB36 Chromosome
HEATHER VALLIERt AND N. E. WELKER*Department of Biochemistry, Molecular Biology, and Cell Biology,
Northwestern University, Evanston, Illinois 60208
Received 12 September 1989/Accepted 30 October 1989
A circular genetic map of Bacillus stearothermophilus NUB36 was constructed by transduction withbacteriophage TP-42C and protoplast fusion. Sixty-four genes were tentatively assigned a cognate Bacillussubtilis gene based on growth response to intermediates or end products of metabolism, cross-feeding,accumulation of intermediates, or their relative order in a linkage group. Although the relative position ofmany genes on the Bacillus stearothermophilus and Bacillus subtilis genetic map appears to be similar, somedifferences were detected. The tentative order of the genes in the Bacillus stearothermophilus aro region isaspB-aroBAFEC-tyrA-hisH-(trp), whereas it is aspB-aroE-tyrA-hisH-(trp)-aroHBF in Bacillus subtilis. The aroA,aroC, and aroG genes in Bacillus subtlis are located in another region. The tentative order of genes in the tipoperon of Bacillus stearothermophilus is trpFCDABE, whereas it is trpABFCDE in Bacillus subtilis.
Organisms adapted to high temperature have probablyevolved unique solutions to the biochemical problems im-posed by this environment. Insights into the mechanisms ofthermophily have been revealed by two types of investiga-tions: (i) comparison of the properties of enzymes and othercell structures of eubacteria and archaebacteria having verydifferent temperature ranges of growth, and (ii) comparisonof the structure or thermostability, or both, of cell constitu-ents of an organism grown at graded temperatures betweenthe minimum and maximum temperature of growth. It isclear from these investigations that the ability to grow at hightemperature cannot be explained by a single mechanism but,rather, that thermophiles appear to use a variety of mecha-nisms. Studies in this field have been hampered because thegenome of a thermophilic bacterial species has not yet beencharacterized.Recent progress in the development of genetic techniques
for Bacillus stearothermophilus NUB36 (4, 14, 15) makes itpossible to elucidate the molecular and genetic mechanismsof thermophily in this organism. The genetic characteriza-tion of the B. stearothermophilus NUB36 genome is the firststep in attaining this goal. In addition, these studies willmake it possible to uncover features that distinguish genomeorganization and function in thermophilic bacilli from that inBacillus subtilis and other mesophilic bacilli. Previous stud-ies (4, 14) indicated that markers on the B. stearothermophi-lus chromosome would exhibit the same phenotype andwould be located in the same relative position as the analo-gous markers on the B. subtilis chromosome. This facilitatedthe selection of markers used in these studies. In this paperwe report on the isolation of a repertoire of mutant strainsand the construction of a genetic map by using transductionand protoplast fusion.
MATERIALS AND METHODSMedia and growth conditions. Cultures were grown in
modified LB medium (4) and minimal glucose (MG) medium(4). Solid LB and MG media contained 1.5% agar, and LB
* Corresponding author.t Present address: Stanford University Medical Center, Stanford,
CA 94305.
top agar contained 0.6% agar. Bacto-Agar (Difco Laborato-ries, Detroit, Mich.) was used in solid MG medium. Whenrequired, MG medium was supplemented with the nutrientsL-alanine, L-arginine, L-aspartate, L-glutamate, L-glycine,DL-homoserine, or L-phenylalanine at 40 jig/ml; L-histidine,L-methionine, or DL-tyrosine at 20 ,ug/ml; L-leucine, L-isoleucine, L-threonine, adenine, hypoxanthine, or uracil at10 pg/ml; guanine, L-serine, L-tryptophan, or L-valine at 5,ug/ml; L-cysteine or riboflavin at 2 ,ugIml; nicotinate at 0.1,ug/ml; and thiamine hydrochloride or biotin at 0.01 ,ug/ml.Resistance mutants were grown in LB medium containingstreptomycin (20 ,g/ml), kasugamycin (2.5 mg/ml), chloram-phenicol, rifampin, tunicamycin (5 ,ug/ml), lincomycin (0.5,ug/ml), sodium azide (0.01 M), or caffeine (5 mg/ml) or MGmedium containing 5-fluorouracil (0.1 ,ug/ml), L-argininehydroxamate (50 pug/ml), or S-(2-aminoethyl)-L-cysteine (25,ug/ml) and 100 jig of L-asparagine per ml (8).
Bacterial strains and bacteriophage. Generalized trans-ducing phage TP-42C was described previously (14). Thebacterial strains used in this study are listed in Table 1. Thephenotypic description of each genetic marker is given inTable 2. Bacterial cultures were grown, and phage TP-42Cwas propagated and assayed as described by Welker (14).
Mutagenesis and isolation of mutants. Mutagenesis byN-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was doneby the procedure described by Chen et al. (4). Auxotrophicmutants were isolated by plating samples of diluted muta-genized cells onto MG plates containing either 0.002%tryptone, 0.004% yeast extract, or 0.001% casein hydroly-sate. Specific auxotrophic mutants were isolated by platingmutagenized cells onto MG medium containing 0.01 ,ug ofthe required nutrient per ml, except for L-aspartate andL-cysteine (0.1 jxg/ml), nicotinate (5 ng/ml), and thiamine andbiotin (0.5 nglml). The auxotrophic mutants were character-ized as described by Chen et al. (4).Mutants resistant to antibiotics, sodium azide, caffeine, or
amino acid and pyrimidine analogs were isolated by platingeither mutagenized cells or exponential-phase cells at highdensity on LB or MG plates containing the compound.Spontaneous sporulation-deficient (Spo-) mutants were iso-lated as described by Welker (14). The reversion frequencyof all the mutations was between 5 x l0e and 7 x 10-8.
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794 VALLIER AND WELKER
TABLE 1. B. stearothermophilus strains used in this study
Strain Characteristics' Origin or referenceb
Wild typethr-ihom-I gly-ihis-imet-irib-iasp-ihom-irfm-i met-2Rif' Hsr- Hsm- Spo-gua4gua-6his-3pur-Spur-6his4leu-2rib-2ser-Iarg-9rib-3met-3gly-4tyr-iilv-2glu-itrp-lmet4arg-13arg-14cys-ipur-21pur-22met-Sgua-iIrfm-i str-i cml-l Spo-
trp-2trp4trp-5asp-3cys-2cys-3azi-2trp-6trp-7trp-13str-3str4tmr-iaro-iaro-2thi-iaro4aro-5aro-7aro-8aro-10aro-12ala-ibio-2nic-2pyr-2pyr-3aec-iaec-2ahr-iahr-2
4444MNNG, NUB3614144MNNG, NUB3618 (4)14MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36Spontaneous,NUB3645 (14)
MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36Spontaneous, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36Spontaneous, NUB36Spontaneous, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36MNNG, NUB36Spontaneous, NUB36Spontaneous, NUB36Spontaneous, NUB36Spontaneous, NUB36
TABLE 1-Continued
Strain Characteristicsa Origin or referenceb
NUB36454 ahr-3 Spontaneous, NUB36NUB36456 ksg-J Spontaneous, NUB36NUB36458 ksg-2 Spontaneous, NUB36NUB36460 lin-i Spontaneous, NUB36NUB36462 caf-i Spontaneous, NUB36NUB36464 fur-i Spontaneous, NUB36NUB36466 aec-3 Spontaneous, NUB36NUB36468 fur-2 Spontaneous, NUB36NUB36470 ksg-3 Spontaneous, NUB36
a Abbreviations: Rif, rifampin resistance; Hsr-, host-specific resistance;Hsm-, host-specific modification; Spo-, cells do not form spores when grownon LB medium at 60°C. The gene symbols (mutant types) are the same asthose used by Piggot and Hoch (10).
b MNNG, Mutagenesis of indicated strain with N-methyl-N'-nitro-N-nitrosoguanidine; Spontaneous, spontaneous mutant isolated from the indi-cated strain.
rfm-J replaces the alternative designation of rif-i (14).
Characterization of auxotrophic mutants. The mutantswere characterized by growth response to intermediates orend products of metabolism. Cross-feeding between trp, aro,and pyr mutants was determined by streaking cells of threemutant strains on an MG plate in close proximity with eachother. The plates were incubated for 48 h at 60°C. A growthresponse between adjacent strains was evidence of cross-feeding between the two strains.Tryptophan mutants were tested for the accumulation of
metabolic intermediates. Mutant cultures were grown in MGmedium containing 0.05% acid-hydrolyzed casein and 1 ,ugof L-tryptophan per ml at 60°C. The cells were removed bycentrifugation, and the supernatant fluids were tested for thepresence of anthranilate (5, 12), 1-(O-carboxyphenyl)-1-deoxyribulose (5, 12), indole-3-glycerolphosphate (16), andindole (13).Mutants having the same phenotype were further sepa-
rated into transduction groups by using transducing phageTP-42C.
Transduction. Transduction was carried out as describedby Welker (14). For transduction of resistance markers, thedonor carried the resistance allele and auxotrophic recipi-ents carried the sensitive allele. The cotransduction fre-quency is the number of transductants carrying the unse-lected marker divided by the total number of transductantstested. The cotransduction frequency for markers that havethe same phenotype was calculated by using the proceduredescribed by Callahan et al. (1).
Protoplast fusion. Protoplast fusion was carried out asdescribed by Chen et al. (4). The auxotrophic fusion parentswere either Stri or Cmlr, and Strr Cmlr recombinants wereselected from the fusion products. Each pair of nonselectedmarkers was assigned a coinheritance frequency (CIF) (4).The CIF for a specific pair of markers is defined as thepercentage of Strr Cmlr recombinants that have either paren-tal phenotype for that specific pair of markers. CIF values of60% or higher were used to establish linkage between twomarkers (4).
RESULTS AND DISCUSSION
Grouping of markers by transduction. Nine linkage groupswere established by using two-factor crosses with trans-ducing phage TP-42C. Data from two-factor crosses arepresented only for linkage group A (Table 3). Cotransduction
NUB36NUB361NUB362NUB363NUB368NUB369NUB3611NUB3625NUB36105CNUB36106NUB36110NUB36127NUB36182NUB36183NUB36184NUB36185NUB36187NUB36192NUB36193NUB36212NUB36226NUB36229NUB36230NUB36231NUB36235NUB36240NUB36248NUB36251NUB36253NUB36256NUB36257NUB36262NUB36263NUB36269NUB36285NUB36321C
NUB36328NUB36330NUB36331NUB36333NUB36335NUB36336NUB36341NUB36342NUB36343NUB36349NUB36360NUB36361NUB36365NUB36375NUB36376NUB36381NUB36382NUB36383NUB36385NUB36386NUB36388NUB36390NUB36424NUB36427NUB36429NUB36433NUB36434NUB36446NUB36448NUB36450NUB36452
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GENETIC MAP OF THE B. STEAROTHERMOPHILUS CHROMOSOME 795
TABLE 2. Genetic markers of B. stearothermophilus NUB36Marker Phenotypea
aec-i, aec-2,b aec-3.......Resistance to S-(2-aminoethyl)-L-cysteineahr-i, ahr-2, ahr-3 ........Resistance to arginine hydroxamateala-i ............. Ala requirementarg-9, arg-14 ............. Arg, Om, or Cit requirementarg-13 ............. Arg requirementaro-i, aro-2, aro4, Shi or Chr or Phe, Tyr, and Trp
aro-5 ............. requirementaro-7, aro-8, aro-JO .......Chr or Phe, Tyr, and Trp requirementaro-12 .............. Phe or Tyr requirementasp-i, asp-3 ............. Asp requirementazi-2 .............. Resistance to sodium azidebio-2 .............. Bio requirementcaf-I .............. Resistance to caffeinecml-l .............. Resistance to chloramphenicolcys-i .............. Cys, Met, Cth, Sfd, or Sft requirementcys-2 ............. Cys, Met, Cth, Hcy, or Sfd requirementcys-3 ............. Cys requirementfur-i, fur-2 .............. Resistance to 5-fluorouracilglu-i ............. Glu or Asp requirementgly-ic .............. Gly or Ser requirementgly4............. Gly requirementgua4, gua-6, gua-ii .....Gua requirementhis-i, his-3, his4 ..........His requirementhom-i............. Hom or Thr and Met requirementilv-2 .............. Ile and Val requirementksg-i, ksg-2, ksg-3 ........Resistance to kasugamycinleu-2 .............. Leu requirementlin-i ............. Resistance to lincomycinmet-i, met-2 .............. Met, Hcy, Cth, Cys, Sfd, or Sft
requirementmet-3, met-S ............. Met, Hcy, or Cth requirementmet4.............. Met requirementnic-2 .............. Nic requirementpur-5 .............. Ade or Gua requirementpur-6 .............. Ade requirementpur-21 ............. Gua or Hpx requirementpur-22.............. Ade, Gua, or Hpx requirementpyr-2 .............. Cbp, Cba, Oro, Thy, Ura, or Cyt
requirementpyr-3 .............. Cba, Oro, Thy, Ura, or Cyt requirementrfm- . .............Resistance to rifampinrib-i, rib-2, rib-3 ...........Rib requirementser-i ............. Ser requirementstr-i, str-3, str4 ...........Resistance to streptomycinthi-i .............. Thi requirementthr-i .............. Thr requirementtmr-i .............. Resistance to tunicamycintrp-i, trp-6, trp-7,
trp-13 ............. Trp or Ind requirementtrp-2, trp-5 ............. Trp requirementtrp4 ............. Trp, Ind, or Ant requirementtyr-i .............. Tyr requirement
a Abbreviations: Ade, adenine; Ala, alanine; Ant, anthranilate; Arg, argin-ine; Aro, aromatic; Asp, Aspartate; Bio, biotin; Caf, caffeine; Cba, N-carbamyl-DL-aspartate; Cbp, carbamyl phosphate; Chr, chorismate; Cit,citrulline; Cth, cystathionine; Cys, cysteine; Cyt, cytosine; Fur, 5-fluorou-racdil; Glu, glutamate; Gly, glycine; Gua, guanine; His, histidine; Hom,homoserine; Hcy, homocysteine; Hpx, hypoxanthine; Ile, isoleucine; Ind,indole; Leu, leucine; Met, methionine; Nic, nicotinate; Orn, ornithine; Oro,orotate; Phe, phenylalanine; Pur, purine; Pyr, pyrimidine; Rib, riboflavin;Ser, serine; Sfd, sulfide; Sft, sulfite, Shi, shikimate; Thi, thiamine; Thr,threonine; Trp, tryptophan; tyr, tyrosine; Ura, uracil; Val, valine.
b aec-2 cells are less resistant to S-(2-aminoethyl)-L-cysteine when threo-nine is added to MG medium.
c Colonies of gly-) on MG medium containing 5 .ug of serine per ml weresmall, but were normal size when both serine (5 pLg/ml) and threonine (20,ug/ml) were added to MG medium
TABLE 3. Cotransduction of markers in the region from met4 totrp-2 in the B. stearothermophilus chromosomea
Donor Recipient Cotransduction Distance betweengenotype genotype (%)b markersc
met4 asp-i 1/223 (1%) 99met-3 asp-i 48/170 (28%) 72his-i asp-i 5/155 (3%) 97his-i met-3 0/250his-i met4 0/283his-i tyr-i 232/249 (93%) 7his-i trp-l 40/123 (35%) 65his-i trp-2 16/160 (8%) 92his-i trp4 0/316his-i trp-6 38/94 (40%) 60his-i trp-7 34/142 (24%) 76his-i trp-13 117/176 (67%) 33his-i gly-i 0/270his-i gly4 0/250his-i aro-i 74/112 (66%) 34his-i aro-2 140/240 (58%) 42his-i aro4 156/193 (81%) 19his-i aro-8 149/204 (73%) 27his-i aro-10 136/195 (70%) 30his-i rib-i 0/250his-i rib-2 0/250trp-i asp-i 0/200trp-l met-3 0/200trp-i met4 0/150trp-l gly-i 0/200trp-l gly4 0/200trp-l ser-i 0/150trp-l aro-i 0/200trp-l aro-2 0/200trp-l aro4 3/250 (1%) 99trp-l aro-8 2/200 (1%) 98trp-l aro-10 0/200trp-l rib-i 0/200trp-l rib-2 0/200a Donor and recipient strains were Spo- and Spo+, respectively.b Fractions represent the number of transductants carrying the donor
(unselected) marker/total number of transductants tested. The values inparentheses represent the percent cotransduction, obtained by multiplying thefraction by 100.
c Distances are expressed as 100 minus the percent cotransduction.
values were obtained from other two-factor crosses (the totalnumber of transductant colonies analyzed is in parentheses),and the combined data were used to construct a map ofeach linkage group (Fig. 1). Genes that are linked but werenot ordered by transduction are indicated by parentheses.The relative order shown for these genes is based on theorder of the putative analogous genes on the B. subtilischromosome.
All the trp genes, except trp4, of linkage group A cotrans-duced with his-l. The relative order of five of the trp geneswas confirmed by two-factor crosses with the trp mutants asboth donors and recipients. The relative positions of tyr-i,the aro genes, and asp-i were determined by crosses withhis-l. Cotransduction was not detected with his-i and met-3or met4. Linkage was observed between trp-i and aro4 andaro-8, but not with aro-10, aro-i, or aro-2.The genes pur-6, gua4, gua-ii, and tmr-i of linkage group
B were not ordered by two-factor crosses. The relativepositions of these genes, however, were verified by proto-plast fusion (Fig. 2). The ksg-3 gene was linked to met-2 (250colonies) but not to met-i (132 colonies), and lin-i was linkedto met-i (191 colonies) but not to met-2 (250 colonies). Thus,the location of ksg-3 and lin-i with respect to met-i and
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796 VALLIER AND WELKER
Group A
I9972
97
- met-4
-rmet-3
asp-I
-aro-2aro- 1
aro- 4-aro-4-tyr-1-his-1
- trp-7trp-13 /
trp-6trp-1:
trp -2
981!5-trp- 13
trp- 1
Ltrp -2trp -4
j(7)
(4)
(9)
97
(5)
E26 45
94
95
,38
Group B
T -pur-6
I
92
56
95
93
-gua-4 94
gua- I1
ksg-3
95
rfm- 1
cml- 1met-2
cys-2
mat. I, .t- I 9 6
79
-str-1-In-i1 2
-(tmr 1)- (aro- 7)
99
.992
23,
74
i
99
99
58
97
65
96
Group C
i -f(blo-2)
(ksg- 1)
(ala-1)hom-i
-thr- 1
-aec-2
- aec- 1-ksg-2
-his-3,4
cys-3
azl-2
met-2 was established by four two-factor crosses. The rela-tive order of aro-7 and tmr-i (245 colonies) was not deter-mined.The genes ksg-2 and aec-i in linkage group C cotrans-
duced with thr-i (250 colonies each), but neither gene wouldcotransduce with hom-i (216 and 250 colonies, respectively).Linkage was established between ksg-i and ala-i (190 colo-nies), hom-i (150 colonies), thr-i (250 colonies), and bio-2(150 colonies); however, the relative order of ksg-i withrespect to bio-2 and ala-i was not established by two-factorcrosses. The genes his-3 and his4 belong to the sametransduction group. However, thr-i cotransduced with his-3(165 colonies) but not his4 (188 colonies), and hom-i did notcotransduce with either his-3 (168 colonies) or his4 (188colonies). The gene azi-2 cotransduced with cys-3 (250colonies) and his4 (500 colonies) but not with his-3 (250colonies). These results indicate that the two his genes lievery close to each other.
The asp-3 gene in linkage group D cotransduced with gly4(245 colonies) but not with ser-i (250 colonies), and gly4was not linked to rib-3 (250 colonies). Thus, the relativeorder of the ser-i, gly4, ahr-i, asp-3, and rib-3 genes wasestablished by two-factor crosses. The gly-i mutant has adifferent phenotype from that of gly4. The gly-i gene liesclose to gly4 based on two-factor crosses with the glymutants as both donors and recipients. Cotransduction val-ues from two-factor crosses positioned rib-2 between ahr-i(250 colonies) and asp-3 (250 colonies); however, linkagewas detected between gly4 and asp-3 (245 colonies) but notbetween gly4 and rib-2 (265 colonies). These results indicatethat rib-2 may lie on the other side of asp-3. The relativeorder of str4 and aro-S (200 colonies) was not determined.The pur genes in linkage group E were ordered by two-
factor crosses with the pur mutants as both donors andrecipients. Althcigh cotransduction was detected betweencaf-i and pur-5 (250 colonies), and not between fur-i and
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GENETIC MAP OF THE B. STEAROTHERMOPHILUS CHROMOSOME 797
Group D
ser- 1gly-1,4
ahr- 14
(rIb-2)
(asa-3)rib-
95
022
91
- (str-4)
Group E
(fur- 1)
(caf-1)
(gua-6)
a pur-5 T T
pur-2i ~(2)1(2)
pur-5 (7)
Group F
T I (str-3)
164154jj
84
88
96
79
- pyr-2- pyr-3
- cys- 1
fur-2
- (aro-5)
Group G
| nic-2
98
91
62
Group H
j |(arg-14)
94
lIv-2
Group I
(met-5)
71
- (ahr-2)
lou-2
- aec-3
pur-5 (250 colonies), thefur-i, caf-i, and gua-6 genes may lieon either side of the pur genes.The order of pyr-2, pyr-3, cys-J, and fur-2 genes (250
colonies of each cross) in linkage group F was determined bytwo-factor crosses. However, the location of str-3 relative tothe other genes in the linkage group was not determined inthis study.
Cotransduction was detected between aec-3 and leu-2 (165colonies) and ilv-2 (250 colonies), leu-2 and ilv-2 (250 colo-nies), and ilv-2 and nic-2 (250 colonies), but not betweenleu-2 and nic-2 (250 colonies). Thus, the relative order of allthe genes in linkage group G was established by two-factorcrosses.The order of arg-14 and ahr-2 (250 colonies) in linkage
group H and met-S and arg-9 (250 colonies) in linkage groupI was not determined.
Ordering linkage groups by protoplast fusion. Chen et al.(4) proposed that protoplast fusion would be an ideal tool for
FIG. 1. Genetic maps of transduction linkage groups A throughH. The arrows by the genetic map (solid lines) point from the donor(unselected marker) to the recipient (selected marker). The numberby the arrows is the genetic distance (Table 3, footnote c) betweenthe two markers. The number within the parentheses by the cross-hatched lines (part of linkage group A ([trp-13 to trp4]) and part oflinkage group E [pur-5 to pur-22]) is the cotransduction frequency(CF), determined as follows: the expected number of prototrophsfor each cross (E) was calculated as E = (MflWd) (Win), where Md isthe number of prototrophs observed when using each trp or purmutant as a donor to a thr recipient, Wd is the number of prototrophsobserved with the wild-type donor crossed to the same recipient,and Wm is the number of prototrophs observed when the wild typewas used as a donor to each trp or pur mutant. CF was determinedas CF = (E - O)IE, where 0 is the number of observed prototrophsin the trp x trp or pur x pur crosses. Markers that are linked buthave not been ordered by transduction analyses are indicated byparentheses.
mapping the B. stearothermophilus chromosome. This pro-posal was based on the assumption that the recombinantsdetected among the fusion products were normal haploidrecombinants. It is possible, however, that the geneticstructure of the fusion recombinants is altered. This wastested by isolating a hom-i thr-i fusion recombinant andusing the double auxotrophic mutant as both a donor and arecipient in transductions with TP-42C. The results areshown in Table 4. The cotransduction values for the hom-iand thr-i genes in the fusion recombinant were the same asthe values obtained for these two genes in Hom- Thr+ orHom+ Thr- strains. These results indicate that the linkagebetween these two genes was not altered in the fusionrecombinant. Thus, protoplast fusion is a reliable techniquefor the ordering of transduction linkage groups.Each fusion parent carried one gene from a transduction
linkage group. In some fusions, one parent carried the glu-igene, which was not linked to any of the other genes by
Ii
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798 VALLIER AND WELKER
15 13I1 s5
38
1 9
420
22
1 9
-pur-6gua-11
.1 met- 1tmr-I
I3 gua-6_pur-5
30 pur-21
met-5
8 arg-9
- - i cys- I
IP glu-i
40
3
asp-I
his-I
-gly-4
asp-3
- ilv-2
- leu-2
Linkage Group B
Linkage Group E
Linkage Group I
Linkage Group F
Linkage Group
Linkage Group
A
D
Linkage Group G
- hom- I
- his-4
pur-6
gua-II
Linkage Group C
Linkage Group B
FIG. 2. Ordering transductional linkage groups by protoplast fusion. The wide solid lines represent transduction linkage groups, andbroken lines represent gaps between neighboring transduction linkage groups. The length of the lines does not represent the distance betweenmarkers or transduction linkage groups. The narrow solid lines indicate fusions between strains carrying the indicated markers. The numbersby the lines are the distances expressed as 100 - CIF (Table 5, footnote b), for pairs of markers located on different transduction linkagegroups. CIF values of 60% of higher were used to establish linkage between two markers.
transduction. At least four two-factor crosses, with parentsthat carry genes from different linkage groups, were used toestablish the order of three linkage groups. CIEs weredetermined for each pair of genes; the combined data areshown in Table 5. The relative order of eight of the ninetransduction linkage groups and the glu-i gene was estab-lished by protoplast fusion (Fig. 2). The location of linkagegroup H was not determined in this study. The pur-6 andgua-ii genes of linkage group B were linked to his4 inlinkage group C, and pur-6 was also linked to gua-6 inlinkage group E. These results establish the genetic circular-ity of the B. stearothermophilus chromosome.
The relative order of pur-6, gua-Ji, met-i, and tmr-i inlinkage group B, gua-6, pur-S, and pur-21 in linkage group E,asp-i and his-i in linkage group A, gly4 and asp-3 in linkagegroup D, and hom-i and his4 in linkage group C establishedby transduction was verified by protoplast fusion.These combined results indicate that protoplast fusion can
be used to order transduction linkage groups, verify theorder of genes in a linkage group, and locate individual geneson the genetic map.Comparison of the genetic map of the B. stearothermopldus
chromosome with the B. subtUis map. By using characteristicssuch as growth response to intermediates or end products of
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GENETIC MAP OF THE B. STEAROTHERMOPHILUS CHROMOSOME 799
TABLE 4. Cotransduction of the Hom and Thr markers ofB. stearothermophilus NUB36
Relevant phenotype of: Cotransduction(%)' with
Donor Recipient phage TP-42C
Hom- Thr+ (fusion Hom+ Thr- (fusion 117/300 (39%)parent) parent)
Hom+ Thr- (fusion Hom- Thr+ (fusion 130/350 (37%)parent) parent)
Hom+ Thr+ (wild Hom- Thr- (fusion 152/400 (38%)type) recombinant)
Hom- Thr- (fusion Hom+ Thr+ (wild 18/48 (38%)recombinant) type)a Factions represent number of transductants carrying the unselected
markers(s)/total number of transductants tested. The values in parenthesesrepresent the percent cotransduction, obtained by multiplying the fraction by100.
metabolism, cross-feeding, or the relative order within alinkage group, each of 64 genes was tentatively assigned acognate B. subtilis gene (Table 6). Genes that were notordered by transduction or protoplast fusion are in paren-theses.The region in linkage group B that contains cys-2, met-i,
TABLE 6. Proposed relationship of B. stearothermophilusmarkers to the analogous markers on the B. subtilis chromosome
B. subtilis chromosomebB. sterothermophilus
marker'arker Map locationAnalogous marker(degrees)Linkage group Bpur-6gua4, gua-iiksg-3rfm-Icml-lcys-2str-Ilin-i(tmr-i)(aro-7)
Linkage group E(fur-i)(caf-1)(gua-6)pur-22
Linkage group H(arg-14)(ahr-2)
purAguaAksgArfm (rpoB)cml (rplO)cymstrA (rpsL)lintmrA or tmrBaroI
355360/0
410101112252
25
furBcafAguaBpurB
argOcahrD
41465055
9899
TABLE 5. Coinheritance frequencies for pairs of markersfrom all fusionsa
Parent no. 1 Parent no. 2
Relevant Transduction Relevant Transduction CIF (%)bmarker linkage group marker linkage group
pur-6 B met-i B 87 (13%)pur-6 B tmr-I B 85 (15%)pur-6 B gua-6 E 62 (38%)pur-6 B pur-21 E 45gua-1i B met-i B 89 (11%)gua-ii B tmr-i B 85 (8%)met-5 I cys-i F 92 (8%)met-5 I glu-Tc 81 (19%)arg-9 I cys-i F 97 (3%)arg-9 I glu-Kc 96 (4%)met-5 I gua-6 E 70 (30%)met-5 I pur-5 E 78 (22%)met-5 I pur-21 E 81 (19%)met-5 I asp-i A 80 (20%)met-5 I his-i A 27met-5 I gly4 D 22met-5 I asp-3 D 20arg-9 I asp-i A 93 (7%)arg-9 I his-i A 91(9%)arg-9 I gly4 D 75 (25%)arg-9 I asp-3 D 61 (39%)ilv-2 G gly4 D 61 (39%)ilv-2 G hom-i C 66 (33%)ilv-2 G his-i A 48ilv-2 G his4 C 29leu-2 G hom-i C 79 (21%)leu-2 H his4 C 64 (36%)pur-6 B his4 C 73 (27%)gua-ii B his4 C 68 (32%)a The number of recombinants analyzed in each fusion varied between 350
and 895. Parental strains were either Cmlr or Strr, and both parental strainswere Rif. Linkage groups are shown in Fig. 1.
b CIF is the percentage of the Strr Cmlr recombinant class that containseither of the parental phenotypes for the indicated pair of markers. Numberswithin parentheses are the relative map distance expressed as 100 minus theCIF (values of 60,% or higher).
C glu-J is not linked to any other markers by transduction.
Linkage group I(met-5)(arg-9)
Linkage group F(str-3)pyr-2pyr-3cys-ifur-2
Linkage group Aasp-iaro-2aro-iaro-10aro-8aro4tyr-ihis-itrp-7, trp-13, trp-6,
trp-i, trp-2, trp4
Linkage group Dser-Igly-igly4ahr-I(str4)rib-3(aro-5)
Linkage group Gnic-2ilv-2leu-2aec-3
Linkage group C(bio-2)(ksg-i)(ala-i)
metAarg-342
strBpyrApyrBcysCfurA
aspBaroBaroAaroFaroEaroCtyrAhisHtrpF, trpC, trpD, trp-A,
trpB, trpE
serserAcglyAahrCstrCribOaroD
nicilvB or ilvCleuA, leuB, or leuCaecA
bioA, bioB, or bio-112ksgBala
Continued or
115115
130135135140140
200210265210210210205205205
210208210215220222230
245250250250
270280280
a following page
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800 VALLIER AND WELKER J. BACTERIOL.
TABLE 6-Continued
B. subtilis chromosomebB. sterothermophilus
ous mra Map locationAnalogous marker (degrees)
hom-i hom 290thr-I thrA 290aec-2 aecB 290ksg-2 ksgC 290his4 hisA 299cys-3 cysB 300azi-2 azi 310
Markers not mappedby transduction
glu-J gltA or gltB 180arg-13 argG or argH 260aro-12 aroG 270a Transduction linkage groups are shown in Fig. 1. Markers that have not
been ordered by transduction are in parentheses."Reference 10.Reference 13.
and met-2 may be similar to the region of the B. subtilischromosome containing cysA, css, hts, and cym. This is acomplex locus that has not been completely characterized(7, 10). The phenotype of cys-2 is analogous to cym in B.subtilis (9), but a mutant that has a phenotype similar tomet-i or met-2 has not been reported for B. subtilis.Three pur genes are located in linkage group E. However,
mutants having a phenotype similar to pur-5 and pur-21 havenot been reported for B. subtilis.The pyr genes were placed into five groups based on
growth response to nutrients and into four groups based oncross-feeding analyses. Two of the pyr genes located inlinkage group F may be similar to pyrA and pyrB of B.subtilis. The locations of the other pyr genes were notdetermined in this study.Linkage group A contains the trp genes and most of the
aro genes. The aro-1, aro-2, aro4, aro-8, and aro-10 mu-tants have a similar phenotype to that of the aroA, aroB,aroC, aroE, and aroF mutants, respectively, of B. subtilis(6). The order of the genes in the B. stearothermophilus aroregion appears to be aspB-aroBAFEC-tyrA-hisIH-(trp). Incontrast, the organization of these genes in B. subtilis isaspB-aroE-tyrA-hisH-(trp)-aroHBF, with aroA, aroC, andaroG located in another region. We did not isolate a B.stearothermophilus mutant that is analogous to an aroHmutant of B. subtilis.The trp mutants were characterized by growth response to
nutrients, cross-feeding analyses, and the accumulation ofmetabolic intermediates (Table 7). The trp-J, trp-2, trp4,trp-6, trp-7, and trp-13 genes may be similar to the trpA,trpB, trpE, trpD, trpF, and trpC genes, respectively, of B.subtilis. The order of the genes in the trp operon of B.stearothermophilus is trpFCDABE, whereas it is trpABFCDE in B. subtilis (2, 3, 17). Callahan et al. (1) reportedthat the relative positions of the trpB, trpC, trpD, and trpEgenes of Bacillus megaterium were similar to the order ofthese genes in B. subtilis. The B. subtilis metB gene islocated next to aspB; however, B. stearothermophilus met-3and met4 mutants each have a phenotype that is differentfrom the metB phenotype.
Additional mutations in the trp and aro regions will beisolated and the proposed order of genes in these regions willbe verified by three-factor transduction and protoplast fusioncrosses.
TABLE 7. Characteristics of trp mutants ofB. stearothermophilus NUB36
GrowthAccumula-Analogous locus
Marker G a tion pro- Cross-feedingb on the B. sub-response' dct tilis chromo-some'
trp4 Trp, Ind, None None trpEor Ant
trp-6 Trp or Ind Ant trp4 trpDtrp-7 Trp or Ind Ant trp4, trp-6 trpFtrp-13 Trp or Ind CDRP trp4, trp-6, trp-7 trpCtrp-J Trp or Ind InGP trp4, trp-6, trp-7, trpA
trp-13trp-2 Trp InGp, Ind trp4, trp-6, trp-7, trpB
trp-13a Abbeviations: CDRP, 1-(O-carboxyphenyl)-l-deoxyribulose; InGP, in-
dole-3-glycerolphosphate; Trp, tryptophan; Ind, indole; Ant, anthranilate.Accumulation of intermediates was determined in the supernatant fluids ofcultures grown in MG medium supplemented with limited amounts of tryp-tophan.
b Each mutant strain in column 1 will feed the mutants on the same line ofcolumn 4.
c Reference 10.
The ahr-J gene of linkage group D may be similar to ahrCofB. subtilis. The B. subtilis ahrC gene is located near aspH.B. stearothermophilus ahr-J is located near asp-3; however,the phenotype of the asp-3 mutant is different from thephenotype ofaspH (constitutive synthesis of aspartase) (10).Two of the three aec genes are in linkage group C. The
aec-2 mutant exhibited a decreased resistance to S-(2-ami-noethyl)-L-cysteine in the presence of threonine. Thus, aec-2may be analogous to aecB of B. subtilis (8). The aec-Jmutant has a different phenotype from that of the aec-2mutant, and aec-J is located next to aec-2. A gene similar toaec-J is not located next to aecB in B. subtilis.We were unable to place glu-J, arg-13, or aro-12 into a
linkage group by transduction. The selection of a cognate B.subtilis gene was based on similarities of the growth re-sponse to nutrients and cross-feeding analyses. The arg-13and aro-12 genes may be similar to the argG or argH andaroG genes, respectively, of B. subtilis. These results indi-cate that arg-13 and aro-12 should map in linkage groups Aand C, respectively. The glu-J gene was mapped by proto-plast fusion.The genetic map of B. stearothermophilus NUB36 may be
similar to the B. subtilis 168 map; however, the identity ofthe genes of B. stearothermophilus with the putative analo-gous genes of B. subtilis must be established by biochemicaland molecular biological techniques.
ACKNOWLEDGMENTS
This investigation was supported by grant DE-FG02-84ER13204from the U.S. Department of Energy.We thank R. Gaber for preparing the genetic maps.
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2. Carlton, B. C. 1966. Fine-structure mapping by transformationin the trp region of Bacillus subtilis. J. Bacteriol. 91:1795-1803.
3. Carlton, B. C. 1967. Transformation mapping of genes control-ling trp biosynthesis in Bacillus subtilis. J. Bacteriol. 94:660-665.
4. Chen, Z., S. F. .Yojcik, and N. E. Welker. 1986. Genetic analysisof Bacillus stearothermophilus by protoplast fusion. J. Bacte-
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1-deoxyribulose, a new compound formed by mutant strains ofAerobacter aerogenes and Escherichia coli blocked in thebiosynthesis of tryptophan. Biochem. J. 72:586-596.
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7. Kane, J. F., R. L. Goode, and J. Wainscott. 1975. Multiplemutations in cysA14 mutants of Bacillus subtilis. J. Bacteriol.121:204-211.
8. Mattioli, R., M. Bazzicalupo, G. Federici, E. Gallori, and M.Polsinelli. 1979. Characterization of mutants of Bacillus subtilisresistant to S-(2-aminoethyl)-L-cysteine. J. Gen. Microbiol. 114:223-225.
9. Piggot, P. J. 1975. Characterization of a cym mutant of Bacillussubtilis. J. Gen. Microbiol. 89:371-374.
10. Piggot, P. J., and J. A. Hoch. 1985. Revised genetic linkage mapof Bacillus subtilis. Microbiol. Rev. 49:158-179.
11. Seeley, H. W., and P. J. VanDemark. 1972. Enzymatic reac-tions, p. 91-92. In D. Kennedy and R. B. Park (ed.), Microbesin action: a laboratory manual of microbiology, 2nd ed. W. H.Freeman and Co., San Francisco.
12. Smith, 0. H., and C. Yanofsky. 1960. 1-(O-Carboxyphenyl-amino)-l-deoxyribulose-5-phosphate, a new intermediate in thebiosynthesis of tryptophan. J. Biol. Chem. 235:2051-2057.
13. Vandeyar, M. A., and S. A. Zahler. 1986. Chromosomal inser-tions of Tn917 in Bacillus subtilis. J. Bacteriol. 167:530-534.
14. Welker, N. E. 1988. Transduction in Bacillus stearothermophi-lus J. Bacteriol. 170:3761-3764.
15. Wu, L., and N. E. Welker. 1989. Protoplast transformation ofBacillus stearothermophilus NUB36 by plasmid DNA. J. Gen.Microbiol. 135:1315-1324.
16. Yanofsky, C. 1956. The enzymatic conversion of anthranilic acidto indole. J. Biol. Chem. 223:171-184.
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