JOURNAL oF BACTEROLOGY, July 1972, p. 73-79 Copyright 0 1972 American Society for Microbiology Vol. 111, No. 1 Printed in U.S.A. Mapping of rod Mutants of Bacillus subtilis D. KARAMATA, M. McCONNELL, AND H. J. ROGERS National Institute for Medical Research, Mill Hill, London, NW7, England Received for publication 25 February 1972 Nine class A salt-dependent rod mutants were mapped on the Bacillus sub- tilis genome by PBS1-mediated transduction. They are distributed into two small linkage groups designated rod B and rod C; mutations in rod B are over 80% cotransducible with pheA and different mutations in rod C are 12 to 21% cotransducible with hisA. It is established that neither rod B nor rod C is linked by transformation to the other identified rod mutations present in 168- ts-200B and 8332 glu-. It is hypothesized that salt-dependent mutations are due to enzyme alterations which are corrected by high salt concentrations. Morphological mutants of Bacillus subtilis, designated rod mutants, grow under restrictive conditions as irregular cocci or round cells, and under nonrestrictive conditions as normal rods (12, 13). The thirteen rod mutants isolated in our laboratory were divided into two classes (13). Class A consists of nine salt-dependent mutants, the morphology of which is corrected by high salt concentration. Class B consists of four mutants which grow as round cells inde- pendently of the salt concentration and are corrected only in very rich media. A tempera- ture-sensitive rod mutant, 168ts-200B, which exhibits the round phenotype at 45 C, has also been described (1, 5), and more recently the isolation of two further rod mutants, which show the round phenotype at 45 C in low salt, has been reported (D. Brooks, and F. E. Young, Bacteriol. Proc., 1971, p. 24). The latter three temperature-sensitive rod mutations have been approximately located on the B. subtilis genome; 168ts-200B is 60% cotransduc- ible with the hisA marker (R. J. Boylan, N. H. Mendelson, D. Brooks, and F. E. Young, Bac- teriol. Proc., 1971, p. 24), whereas the other two mutants seem to be only loosely linked to hisA (D. Brooks and F. E. Young, Bacteriol. Proc., 1971, p. 24). To simplify the physiolog- ical and biochemical analysis of different rod mutants it would be of great interest to estab- lish whether there is any genetic relationship between them. In this paper we have examined the linkage among class A salt-dependent rod mutants, localized these on the genome of B. subtilis, and examined their genetic relationship to other rod mutants. Results pertinent to the nature of salt-dependent mutations are dis- cussed. MATERIALS AND METHODS Bacterial strains. Strains used are listed in Table 1. Media. (i) LS medium was as follows: NH4Cl, 0.535 g; KH2PO4, 0.068 g; Na2SO4, 0.106 g; NH; NO3, 0.096 g; MgCl,, 0.004 g; MnCl2, 0.013 g; Fe- Cl2, 0.46 mg; agar (Difco), 15 g; distilled water, 1 li- ter. Glucose, to a final concentration of 0.5%, and appropriate growth factors were added after sterili- zation. Amino acids were added at the final concen- tration of 50 gg/ml. (ii) T-S medium was as de- scribed by Karamata and Gross (10). When required, media were supplemented with different amino acids or bases to a final concentration of 50 Ag/ml. Transduction. Stocks of phage PBS1 were pre- pared and the transduction was carried out as de- scribed by Karamata and Gross (10). All crosses were made at 37 C. Transformation. Deoxyribonucleic acid (DNA) extraction and preparation of competent cells and crosses were carried out essentially as described by Karamata and Gross (10). Crosses were carried out at 37 C. Selection and screening of recombinants. Auxo- trophic recombinants were selected on appropriately supplemented T-S plates incubated at 35 C. ts+ re- combinants of rod mutants were selected on T-S or LS plates (supplemented with 0.01% sodium gluta- mate). The plates were first incubated for 4 to 6 hr at 35 C and then transferred to 48 C. Auxotrophic or ts+ recombinants were purified by streaking for single colonies before further analysis. They were then screened for other auxotrophic or ts characters by replica-plating, and for rod character by replica- plating onto LS plates and by examining single colo- nies by phase-contrast microscopy. Criteria of genetic linkage and ordering of markers. Recombination index, cotransfer index, and the ordering of markers were determined as de- scribed by Karamata and Gross (10). 73 on February 13, 2020 by guest http://jb.asm.org/ Downloaded from
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JOURNAL oF BACTEROLOGY, July 1972, p. 73-79Copyright 0 1972 American Society for Microbiology
Vol. 111, No. 1Printed in U.S.A.
Mapping of rod Mutants of Bacillus subtilisD. KARAMATA, M. McCONNELL, AND H. J. ROGERS
National Institute for Medical Research, Mill Hill, London, NW7, England
Received for publication 25 February 1972
Nine class A salt-dependent rod mutants were mapped on the Bacillus sub-tilis genome by PBS1-mediated transduction. They are distributed into twosmall linkage groups designated rod B and rod C; mutations in rod B are over80% cotransducible with pheA and different mutations in rod C are 12 to 21%cotransducible with hisA. It is established that neither rod B nor rod C islinked by transformation to the other identified rod mutations present in 168-ts-200B and 8332 glu-. It is hypothesized that salt-dependent mutations aredue to enzyme alterations which are corrected by high salt concentrations.
Morphological mutants of Bacillus subtilis,designated rod mutants, grow under restrictiveconditions as irregular cocci or round cells, andunder nonrestrictive conditions as normal rods(12, 13). The thirteen rod mutants isolated inour laboratory were divided into two classes(13). Class A consists of nine salt-dependentmutants, the morphology of which is correctedby high salt concentration. Class B consists offour mutants which grow as round cells inde-pendently of the salt concentration and arecorrected only in very rich media. A tempera-ture-sensitive rod mutant, 168ts-200B, whichexhibits the round phenotype at 45 C, has alsobeen described (1, 5), and more recently theisolation of two further rod mutants, whichshow the round phenotype at 45 C in low salt,has been reported (D. Brooks, and F. E.Young, Bacteriol. Proc., 1971, p. 24). The latterthree temperature-sensitive rod mutationshave been approximately located on the B.subtilis genome; 168ts-200B is 60% cotransduc-ible with the hisA marker (R. J. Boylan, N. H.Mendelson, D. Brooks, and F. E. Young, Bac-teriol. Proc., 1971, p. 24), whereas the othertwo mutants seem to be only loosely linked tohisA (D. Brooks and F. E. Young, Bacteriol.Proc., 1971, p. 24). To simplify the physiolog-ical and biochemical analysis of different rodmutants it would be of great interest to estab-lish whether there is any genetic relationshipbetween them.
In this paper we have examined the linkageamong class A salt-dependent rod mutants,localized these on the genome of B. subtilis,and examined their genetic relationship toother rod mutants. Results pertinent to thenature of salt-dependent mutations are dis-
cussed.
MATERIALS AND METHODSBacterial strains. Strains used are listed in
Table 1.Media. (i) LS medium was as follows: NH4Cl,
0.535 g; KH2PO4, 0.068 g; Na2SO4, 0.106 g; NH;NO3, 0.096 g; MgCl,, 0.004 g; MnCl2, 0.013 g; Fe-Cl2, 0.46 mg; agar (Difco), 15 g; distilled water, 1 li-ter. Glucose, to a final concentration of 0.5%, andappropriate growth factors were added after sterili-zation. Amino acids were added at the final concen-tration of 50 gg/ml. (ii) T-S medium was as de-scribed by Karamata and Gross (10). When required,media were supplemented with different aminoacids or bases to a final concentration of 50 Ag/ml.
Transduction. Stocks of phage PBS1 were pre-pared and the transduction was carried out as de-scribed by Karamata and Gross (10). All crosses weremade at 37 C.
Transformation. Deoxyribonucleic acid (DNA)extraction and preparation of competent cells andcrosses were carried out essentially as described byKaramata and Gross (10). Crosses were carried outat 37 C.
Selection and screening of recombinants. Auxo-trophic recombinants were selected on appropriatelysupplemented T-S plates incubated at 35 C. ts+ re-combinants of rod mutants were selected on T-S orLS plates (supplemented with 0.01% sodium gluta-mate). The plates were first incubated for 4 to 6 hrat 35 C and then transferred to 48 C. Auxotrophic orts+ recombinants were purified by streaking forsingle colonies before further analysis. They werethen screened for other auxotrophic or ts charactersby replica-plating, and for rod character by replica-plating onto LS plates and by examining single colo-nies by phase-contrast microscopy.
Criteria of genetic linkage and ordering ofmarkers. Recombination index, cotransfer index,and the ordering of markers were determined as de-scribed by Karamata and Gross (10).
tion rod-7c)(13)(13)(13)(13)(13)Derived by introducingby transformation therodBI marker intoBD54 and selecting aleu-8, rodBl recombi-nant
Derived by introducingrodCl in BD54, asabove
Derived by introducingby transformation therodBl marker fromROD4 into BD54 andselecting a proto-trophic rodBI recom-binant
(6)
(6)
(6)(6)
(6)
(6)
(6)(6)
(10)
(16)(2)(1)Derived by introducingby transformationthe tag-i markerinto BD54 and se-lecting a leu-8,metB5, ile+ recom-binant
Derived from a nitro-soguanidine-inducedglu mutant of E10(10). The glu markerwas introduced inBD54 by transforma-tion and a met', leu-8, ile-1 recombinantwas selected
RESULTSSeparation of class A rod mutants into
two subclasses. Before genetic analysis thesalt-dependent class A mutants (13) were di-vided according to their phenotypes into twosubclasses designated Al and A2 (Table 2).Subclass Al consists of four mutants whichgrow as round cells on LS medium containing0.1% Casamino Acids and as normal rods onLS medium supplemented with 0.5% sodiumglutamate. None of these four mutants growsat 48 C on LS plates supplemented with 0.01%sodium glutamate. The temperature-sensitivephenotype is reversed by either 0.8 M NaCl or0.5% glutamate. Subclass A2 consists of fivemutants. These grow as a heterogeneous popu-lation of rod, oval, and round cells on LS me-dium containing 0.1% casein hydrolysate andchange only quantitatively when the mediumis supplemented with 0.5% sodium glutamate;the proportion of rod cells becomes slightlylarger, but the populations remain heteroge-neous. Subclass A2 mutants grow, though veryslowly, at 48 C on LS medium containing0.01% sodium glutamate. Their morphology iscorrected by 0.8 M NaCl.Location of rod markers of ROD4 and
ROD 13 on the B. subtilis genetic map.ROD4 and ROD13 were chosen as representa-tives of subclasses Al and A2, respectively,and the rod mutations were mapped on the B.subtilis genome by phage' PBS1-mediatedtransduction. PBS1 stocks were prepared onstrains ROD204, rodBi (which is a prototroph)and ROD113, rodCl, leu-8, crossed to variousauxotrophic strains listed in Table 1, andplated on appropriately supplemented T-Smedium. Prototrophic recombinants were rep-licated on LS plates containing appropriatesupplements and screened for inheritance ofthe rod character. Results (Table 3) show thatthe rodBI marker is over 80% cotransduciblewith pheA12 and 63% cotransducible with leu-8. rodCI is 17% cotransducible with hisAl, andonly very loosely linked to cysB3, suggestingthat it is situated to the right of hisA (Fig. 1).More evidence about the location of rodmarkers was obtained from three-factor crosses(Table 4). Segregation patterns of variousmarkers are compatible with the above obser-vations and suggest that rodCl and rodBI areordered as follows: cysB3, hisAl, rodCi andleu-8, rodBi, pheA12. Thus we established thatthe rod markers of ROD4 (representative ofsubclass Al) and ROD13 (representative ofsubclass A2) map in two unlinked loci whichwe will designate rodBi and rodCl, respec-tively (Fig. 1).Distribution of other Class A rod mutants
a Mutants were plated so as to obtain single colonies on appropriately supplemented plates which wereincubated at the indicated temperature for 24 hr. About a dozen colonies from each plate grown at 35 C wereexamined for cell morphology with a phase-contrast microscope.
TABLE 3. Linkage of rod markers rodBl and rodCl to auxotrophic markers of known locationa
No. of re-
Total no. combinantsSelected of recoin- inheriting CotransferDonor Recipient marker of unselected index
aPBS1 stocks were prepared on ROD204, rodBl and ROD113, leu-8, rodCl and crossed to the listed auxo-trophic strains. Prototrophic recombinants were selected on appropriately supplemented T-S plates. Trans-ductants were then restreaked for single colonies onto appropriately supplemented LS plates and screenedwith a microscope for the inheritance of the rod character.
Origin cvsB hisA argC leu pheA
1 I' 1.smo rodCl rodBl
FIG. 1. Approximate positions of rodBl and rodCl and smo markers on the Bacillus subtilis genetic map.The mutations were located on the B. subtilis genetic map (6) by PBS1-mediated transduction. The positionsare derived from crosses described in Tables 3, 4 and 8.
into linkage groups. To determine whethersubclass Al mutants form a linkage group, wecrossed by transformation ROD4, trp-2, rodBl;ROD7, trp-2, rodB2; £-OD10, trp-2, rodB3;and ROD11, trp-2, rodB4 to ROD104, leu-8,rodBl and calculated the recombination indexfrom each cross. Results (Table 5) show thatthese mutants form a linkage group designatedB. Although the mutants are closely linked,two out of three crosses yielded ts+ recombi-nants, suggesting that all the mutations are
not located at the same site. By comparing thelargest recombination index between two rodB mutants, which is 0.075, to that of the mostdistant markers of the tryptophan synthetase
gene of B. subtilis, which is 0.2 (3), we esti-mate that rod B mutants almost certainly mapin one gene. Since there is no way of selectingrod+ recombinants from crosses involving sub-class A2 mutants, which grow at high tempera-ture, their linkage to rodCl was examined bymapping them on the B. subtilis genome. Theywere located by three-factor crosses, mediatedby PBS1 transduction, similar to the cross bywhich rodCl was mapped (see Table 4). Re-sults presented in Table 6 show that all sub-class A2 mutants are linked to hisA and sug-gest that they all map in the same region as
rodCl, which is to the right of hisA. Thislinkage group will be designated C. Again the
a PBS1 stocks were prepared on BD40 and crossed with ROD104, and leu+ recombinants were selected onappropriately supplemented T-S plates. Ieu+ transductants were streaked for single colonies, screened forinheritance of phe by replica-plating and for inheritance of rod character by restreaking on LS plates andexamining the cells with a microscope. Similarly, PBS1 stocks prepared on ROD113 were crossed to BD92,and the cys+ recombinants were repurified and screened for inheritance of his and rod characters. The orderof markers was determined according to Hayes (9).
TABLE 5. Linkage between subclass Al rod mutantsa
DonorRecipient Transformants/ml RecombinationDonor Recipient+ od index
a Donor DNA was extracted from mutants ROD4, ROD7, ROD10, and RODll which are rod and leu+. Thewild-type donor DNA was prepared from strain 168 which is rod+ and leu+. The recipient strain was ROD104which is rod and leu. Each donor was crossed to ROD104 by transformation; ts+ recombinants were selectedby plating on LS medium, preincubating the plates for 4 to 6 hr at 35 C and then shifting to 48 C. rod+ shapeof a representative sample (about 100) of the ts+ recombinants from each cross was further confirmed byexamining with a microscope. Ieu+ recombinants were selected by plating on T-S medium supplementedwith tryptophan. The recombination index was calculated as described by Karamata and Gross (10).
TABLE 6. Location of subclass A2 mutants on the Bacillus subtilis genomea
No. of recombinants Total Cotrans-
Donor Recipient Selected h his+ his+ his+ his+ fer indexmarke
cy5-YS cys cys+ exam- hisA- ugse rerod+ rod+ rod- rod- med rod
a Three-factor crosses were carried out by PBS1-mediated transduction. PBS1 stocks were prepared on allsubclass A2 rod mutants and crossed to BD92. his+ recombinants were selected on appropriately supple-mented T-S plates. About 100 recombinants from each cross were restreaked for single colonies on identicalT-S medium and screened for the inheritance of the cys marker by replica-plating on approximately supple-mented T-S plates. Screening for the inheritance of the rod+ marker was done microscopically on coloniesrestreaked on LS medium. The order of markers and the cotransfer index were determined according toHayes (9).
results suggest that all the mutations are lo-cated in a small region but not at the samesite.
Positions of morphological mutants 168ts-200B and 8332 relative to group B and groupC rod mutants. Since the mutation tag-i ofthe temperature-sensitive rod mutant 168ts-200B is reported to be located to the right ofhisA and 60% cotransducible with that marker(R. J. Boylan, N. H. Mendelson, D. Brooks,and F. E. Young, Bacteriol. Proc., 1971, p. 24),we examined whether tag-i and group C rodmutations were cotransformable. The lattermutations were located in the same region byPBS1-mediated transduction (see above).DNA from ROD113, rodCl, leu-8 was preparedand crossed to 172ts-200B, leu-8, metB5, tag-i.ts+ transformants were selected and screenedfor the inheritance of the rod C marker. All102 ts+ colonies examined were found to berod+. This result shows that tag-1 and rodCIare not cotransferable by transformation andconfirms the transduction data which suggestthat tag-1 is much more closely linked to hisAthan is rodCI.Mutant 8332 is a glutamate-requiring auxo-
troph. Its cells grow as normal rods on T-Splates, which contain 0.5% glutamate, andwhen replicated on LS plates they exhibit theround phenotype during the limited period ofgrowth (Karamata and Rogers, unpublisheddata). The round phenotype is not reversibleby 0.8 M NaCl. We determined the position ofthe marker glu-1 relative to other rod markersby examining whether glu-1 was cotransduc-ible with the auxotrophic markers hisAl andpheA12 to which rod C and rod B markers arelinked, respectively. Results presented in Table7 show that glu-1 is cotransducible with nei-ther hisA nor pheA. Therefore this mutant be-longs to yet another linkage group of rod mu-
tants.The above analysis shows that the tempera-
ture-sensitive or salt-dependent or glutamate-
requiring mutants which exhibit the roundphenotype are distributed in four linkagegroups represented by the following markers:tag-i, rodBI, rodCi, and glu-i.Remarks on the "shiny" appearance of
colonies of originally isolated rod mutants.Nearly all of the original rod strains had a
shiny colony appearance on LS plates con-
taining 0.1% casein hydrolysate (or LS + 0.8 MNaCl) as opposed to the "rough" colonies ofthe parent strain 168. To determine whetherthe shininess, due possibly to modifications ofthe cell surface, was related to the rod muta-tions, we examined the segregation of rodBIand the "shiny" colony character in the fol-lowing back-cross. BD54 leu-8, metB5, ile-iwas transformed by ROD4, rodBl, trp-2 DNA;the leu+ recombinants were selected andscreened for the inheritance of rod and "shiny"characters. The two markers segregated inde-pendently, thus establishing that the "shiny"character, designated smo-4 (smo for"smooth"), was due to a mutation differentfrom rodBl. However, microscopical observa-tions revealed that smo-4 strains also had a
slightly abnormal morphology when grown on
LS plates; they usually grow as long chains ofsmall, oval cells. The smo markers of ROD4and ROD13, designated smo-4 and smo-5,were located on the B. subtilis genetic map bycotransduction with auxotrophic markers ofknown location. Results (Table 8) suggest thatboth smo-4 and smo-5 map between cysB andhisA. Therefore the shiny character seems tobe related to the "smo" and "rou" charactersdescribed by Young et al. (17) and Grant andSimon (8), which map in the same segment.We would like to point out that, on LS me-
dium containing 0.1% casein hydrolysate,strains bearing the rodBl and rodCl mutationsstill grew as slightly shiny colonies, distin-guishable from the wild type.By reconstruction experiments we deter-
mined that double mutants rod, smo grow less
TABLE 7. Relative position of markers rodB, rodC, and glu-ja
No. of g1u+recombinants
Total no. Tested for which have CotransferDonor Recipient of glu+ cotransduc- inherited index
a PBS1 stocks prepared on BD92, trp-2, hisA, cysB and BD40, argA, pheA were crossed to 8332, glu-1. Theglu+ recombinants, selected on appropriately supplemented T-S plates, were screened for the inheritance ofthe hisA or the phe marker by replica-plating.
a Donor PBS1 phage was prepared on strains ROD4 and ROD13 and crossed to BD92. cys+ and his+ re-combinants from each cross were selected separately on appropriately supplemented T-S plates and visuallyscreened for the inheritance of the smo character.
well on LS plates than single rod, smo+ mu-tants, and this may be the reason for pickingpreferentially rod-, smo- mutants; rod, smo+mutants grew well and did not appear salt-dependent during the isolation. The doublemutants rod, smo might have arisen originallyfrom two sources: induction of rod mutationsin smo bacteria already present in the popula-ttion (smo mutants arise spontaneously withrelatively high frequencies), or induction ofdouble smo, rod mutants during the treatmentby the mutagen.
DISCUSSIONThe rod mutants 168ts-200B, 8332, and class
A mutants of B. subtilis are distributed intofour genetic linkage groups. Some rod muta-tions are now known to have wall deficiencieswhen grown under nonpermissive conditions.When grown at 45 C, mutant 168ts-200B andthe temperature-sensitive, salt-dependentmutants of Brooks and Young (Bacteriol.Proc., 1971, p. 24) contain either reducedamounts of teichoic acid or poorly glycosylatedteichoic acid in their walls (R. J. Boylan. N. H.Mendelson, D. Brooks, and F. E. Young, Bac-teriol. Proc., 1971, p. 24; D. Brooks, and F. E.Young, Bacteriol. Proc., 1971, p. 24). Thus,modifications in the amount or structure ofteichoic acids are correlated with the roundphenotype. The above mutations have all beenlocated to the right of hisA where the rod Cmutants are also located. Preliminary obser-vations show the rod C mutants also have areduced amount of teichoic acid in their wallswhen grown as cocci in LS medium (McCon-nell, Rogers, and Karamata, unpublisheddata). This suggests that all the rod mutationslocated near hisA may be involved in aspectsof teichoic acid biosynthesis. The round phe-notype of mutants bearing rod B, located in adifferent region of the chromosome, may haveanother cause because one of them, ROD4, is
not deficient in wall phosphorus or glucose andis therefore presumably not deficient in totalamount of teichoic acid (14). The phenotype ofthis group of mutants is reversed by the addi-tion of 0.2% glutamate or glutamine to LS me-dium (see Table 2). At 48 C in LS medium, rodB mutants are glu auxotrophs and in this re-spect resemble other glu auxotrophs such as8332. The latter is one of 36 glutamate-requir-ing mutants, many of which show abnormalmorphology ranging from oval to round cellswhen deprived of glutamate (Karamata andRogers, unpublished data). Preliminary obser-vations suggest that these mutants are distrib-uted in several genes.The above analysis suggests that a rather
large number of mutations can produce around phenotype, and one would thereforeexpect the salt-dependent rod mutants ana-lyzed here to be randomly distributed. How-ever, all four group B rod mutants are veryclosely linked, and the five group C mutantsare clustered in a small linkage group which isnot linked to the tag-i marker as determinedby transformation. The clustering of the muta-tions cannot be accounted for by anomalies inmutant selection since each mutant comesfrom a separate batch of mutagenized cells.Neither can it be due to any specificity of themutagen. Firstly there is ample evidence thatnitrosoguanidine induces mutations at thegrowing point (4) which, in an exponentiallygrowing population, is nearly randomly dis-tributed over the whole genome. Secondly,mutations within each group are not located atthe same site. The strikingly nonrandom dis-tribution of salt-dependent mutants could beexplained by assuming that they are due tomissense mutations producing enzymes withincorrect tertiary structure which can onlyfunction in high salt concentrations. That theintracellular salt concentration in Escherichiacoli can be altered has been shown; the intra-cellular Na+ concentration is proportional to
the external salt concentration and that of K+to the osmolarity of the medium (7). The highprobability of the occurrence of these muta-tions in a few genes may have an explanationsimilar to that for the uneven distribution ofthe temperature-sensitive mutants (10),namely that temperature-sensitive phenotypesoccur with different frequency in differentenzymes. In some proteins, many differentamino acid substitutions may result in a salt-sensitive product, whereas in others the resultmay be an inactive or non-salt-dependentproduct. Such an explanation would demandthe presence of a salt-dependent enzyme in-volved in glutamate or glutamine metabolismin rod B mutants. These metabolites wouldthen reverse the phenotype because the en-zyme is no longer required. Other mutations inB. subtilis, giving rise to proteins apparentlyfunctional in either high salt or at low temper-atures, have been described. The rod mutantsisolated by Brooks and Young (Bacteriol.Proc., 1971, p. 24) are corrected by high salt orlow temperature. Also the phenotype of somegroup B temperature-sensitive DNA mutantsof E. coli is not expressed in media containing0.35 M NaCl (15), suggesting that high saltconcentration allows the ts proteins to func-tion at high temperatures.
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