INTERSPECIFIC TRANSFORMATION IN BACILLUS' JULIUS MARMUR, EDNA SEAMAN, AND JAMES LEVINE2 Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts Received for publication 17 September 1962 ABSTRACT MARMUR, J. (Brandeis University, Waltham, Mass.), E. SEAMAN, AND J. LEVINE. Interspecific transformation in Bacillus. J. Bacteriol. 85:461- 467. 1963.-Deoxyribonucleic acids (DNA) from various species of the taxonomic group Bacillaceae were examined for base composition, ability to carry out interspecific transformation, and formation of molecular hybrids in vitro. The minimal requirement for genetic compatibility among different species and for DNA interaction (both reflecting base sequence homologies) is the similarity of the guanine plus cytosine contents of the DNA. The close correlation between the ability of DNA to be competent in interspecific transformation and to form hybrid molecules on denaturation and annealing provided a rational approach to the study of genetic relationship among organisms for which no genetic exchange has yet been demonstrated. Any or all of the criteria (base composition of DNA, transforma- tion, molecular hybrid formation) can be used as tools in the taxonomic assessment of closely related microorganisms. The transfer of genetic information among microorganisms belonging to different species and genera has been reported. Recombination has been demonstrated among Escherichia coli, Shigella, and Salmonella (Luria and Burrous, 1957; Zinder, 1960a; Miyake and Demerec, 1959; Baron, Carey, and Spilman, 1959). It has also been found that phage-mediated transduction of genetic characters occurs between Escherichia and Shigella (Lennox, 1955; Luria, Adams, and Ting, 1960). Transformation induced by highly polymerized deoxyribonucleic acid (DNA) can I Publication no. 199 from the Graduate De- partment of Biochemistry, Brandeis University. Part of the work was carried out in the Depart- ment of Chemistry, Harvard University. 2 Present address: School of Medicine, Yale University, New Haven, Conn. transfer genetic traits reciprocally among several species of Haemophilus (Leidy, Hahn, and Alexander, 1959; Schaeffer, 1958a) and Neisseria (Catlin, 1960, 1961; Catlin and Cunningham, 1961), and between Streptococcus and Diplococcus pneumoniae (Bracco et al., 1957). In the genetic exchanges by recombination and transduction, the DNA base composition of the members of the group is very similar, or very nearly so (Lee, Wahl, and Barbu, 1956; Belozersky and Spirin, 1960; Lanni, 1960; Sueoka, 1961). These facts would support the suggestion that the efficiency of genetic transfer is a reflection of homologies in the nucleic acids of the related organisms (Schaeffer, 1958b; Zinder, 1960b). The discovery of transformation in Bacillus subtilis led us to ask the following question: could one predict interspecific transformation by comparing the over-all base composition of various Bacillus species to that of B. subtilis? The following hypothesis was tested in this study: only the organisms which have DNA with similar base compositions can be expected to exhibit microhomologies in the structure of their DNA. Two criteria were used for the de- termination of microhomologies: (i) transforma- tion of B. subtilis by high-molecular-weight DNA isolated from other Bacillus species; and (ii) formation of a DNA hybrid band by heating and annealing B. subtilis DNA with DNA of other Bacillus species. MATERIALS AND METHODS Bacterial cultures. The list of organisms em- ployed in this study is presented in Table 1. The cultures were stored in the frozen state in Brain Heart Infusion broth (Difco) containing 10% glycerol. They were cultured in Brain Heart Infusion broth for DNA isolation, or grown overnight on a Tryptose Blood Agar Base (Difco) slant for use in transformation experiments. DNA isolation. DNA isolation was carried out according to the method of Marmur (1961). Cells were harvested at the end of log phase, 461 on May 21, 2021 by guest http://jb.asm.org/ Downloaded from
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INTERSPECIFIC TRANSFORMATION IN BACILLUS'
JULIUS MARMUR, EDNA SEAMAN, AND JAMES LEVINE2
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts
Received for publication 17 September 1962
ABSTRACTMARMUR, J. (Brandeis University, Waltham,
Mass.), E. SEAMAN, AND J. LEVINE. Interspecifictransformation in Bacillus. J. Bacteriol. 85:461-467. 1963.-Deoxyribonucleic acids (DNA) fromvarious species of the taxonomic group Bacillaceaewere examined for base composition, ability tocarry out interspecific transformation, andformation of molecular hybrids in vitro. Theminimal requirement for genetic compatibilityamong different species and for DNA interaction(both reflecting base sequence homologies) is thesimilarity of the guanine plus cytosine contentsof the DNA. The close correlation between theability of DNA to be competent in interspecifictransformation and to form hybrid molecules ondenaturation and annealing provided a rationalapproach to the study of genetic relationshipamong organisms for which no genetic exchangehas yet been demonstrated. Any or all of thecriteria (base composition of DNA, transforma-tion, molecular hybrid formation) can be used astools in the taxonomic assessment of closelyrelated microorganisms.
The transfer of genetic information amongmicroorganisms belonging to different speciesand genera has been reported. Recombinationhas been demonstrated among Escherichia coli,Shigella, and Salmonella (Luria and Burrous,1957; Zinder, 1960a; Miyake and Demerec, 1959;Baron, Carey, and Spilman, 1959). It has alsobeen found that phage-mediated transduction ofgenetic characters occurs between Escherichiaand Shigella (Lennox, 1955; Luria, Adams, andTing, 1960). Transformation induced by highlypolymerized deoxyribonucleic acid (DNA) can
I Publication no. 199 from the Graduate De-partment of Biochemistry, Brandeis University.Part of the work was carried out in the Depart-ment of Chemistry, Harvard University.
2 Present address: School of Medicine, YaleUniversity, New Haven, Conn.
transfer genetic traits reciprocally among severalspecies of Haemophilus (Leidy, Hahn, andAlexander, 1959; Schaeffer, 1958a) and Neisseria(Catlin, 1960, 1961; Catlin and Cunningham,1961), and between Streptococcus and Diplococcuspneumoniae (Bracco et al., 1957). In the geneticexchanges by recombination and transduction,the DNA base composition of the members ofthe group is very similar, or very nearly so(Lee, Wahl, and Barbu, 1956; Belozersky andSpirin, 1960; Lanni, 1960; Sueoka, 1961). Thesefacts would support the suggestion that theefficiency of genetic transfer is a reflection ofhomologies in the nucleic acids of the relatedorganisms (Schaeffer, 1958b; Zinder, 1960b).The discovery of transformation in Bacillus
subtilis led us to ask the following question:could one predict interspecific transformation bycomparing the over-all base composition ofvarious Bacillus species to that of B. subtilis?The following hypothesis was tested in this
study: only the organisms which have DNAwith similar base compositions can be expectedto exhibit microhomologies in the structure oftheir DNA. Two criteria were used for the de-termination of microhomologies: (i) transforma-tion of B. subtilis by high-molecular-weightDNA isolated from other Bacillus species; and(ii) formation of a DNA hybrid band by heatingand annealing B. subtilis DNA with DNA of otherBacillus species.
MATERIALS AND METHODS
Bacterial cultures. The list of organisms em-ployed in this study is presented in Table 1. Thecultures were stored in the frozen state in BrainHeart Infusion broth (Difco) containing 10%glycerol. They were cultured in Brain HeartInfusion broth for DNA isolation, or grownovernight on a Tryptose Blood Agar Base (Difco)slant for use in transformation experiments.DNA isolation. DNA isolation was carried out
according to the method of Marmur (1961).Cells were harvested at the end of log phase,
B. alvei.................................. ATCC 6344 American Type Culture CollectionB. megaterium-cereus ...................... ATCC 14B22 American Type Culture CollectionB. laterosporus............................ ATCC 64 American Type Culture CollectionB. lentus ................................. ATCC 10840 American Type Culture CollectionB. thuringiensis........................... ATCC 10792 American Type Culture CollectionB. circulans .............................. ATCC 4513 American Type Culture CollectionB. sphaericus............................. ATCC 4525 American Type Culture CollectionB. niger .................................. ATCC 6454 American Type Culture CollectionB. subtilis var. niger...................... ATCC 6455 American Type Culture CollectionB. subtilis var. aterrinls.................s.ATCC 6460 American Type Culture CollectionB. firmus ..American Type Culture CollectionB. macerans.............................. ATCC 7069 American Type Culture CollectionB. polymyxa.............................. ATCC 842 American Type Culture Collection
washed, and lysed with lysozyme or sodiumlauryl sulfate. Sodium lauryl sulfate was addedto lysozyme lysates to inhibit deoxyribonucleaseaction. The lysates were deproteinized by re-peated extractions with chloroform-isoamylalcohol. Ribonucleic acid (RNA) was digested byribonuclease treatment. The pure, high-molecular-weight DNA was precipitated withisopropyl alcohol in the presence of 0.3 M sodiumacetate, and dissolved in standard saline-citrate(SSC; 0.15 M NaCl and 0.015 M Na citrate, pH 7).Determination of sedimentation coefficient. The
sedimentation coefficient of S20o w of DNAdissolved in SSC was determined in a Spincoultracentrifuge, model E, at concentrations of 20,g/iml at a speed of 35,600 rev/min, usingultraviolet-absorption optics. The centrifugecell was fitted with a Kel-F centerpiece.
CsCI density gradient centrifugation. Themethod employed was similar to that describedby Meselson, Stahl, and Vinograd (1957). DNAin approximately 5.7 M CsCl was centrifuged at44,770 rev/min for approximately 24 hr, and thebanded DNA photographed by use of ultravioletoptics. By using a standard DNA of knowndensity, the buoyant densities of the bandedDNA were calculated after first tracing the
photographs with a Joyce-Leobel microdensitom-eter.DNA base composition. DNA base composition
was estimated from the melting temperature ofthe absorbance-temperature denaturation pro-files. All DNA samples were heated in SSC at aconcentration of 20 ,g/ml.
Bacterial transformation. Transformation ex-periments were performed, using the method ofSpizizen (1959). The following genetic markerswere used in transformation: ind- ind+(indole); arg -* arg+ (arginine); and Es -* ER(erythromycin). Transformation assays werecarried out at saturating levels of DNA, andcomparisons made at equivalent concentrationsof DNA.
RESULTS
The transformation of B. subtilis by variousBacillus species is listed in Table 2. The DNAbase composition, as well as the sedimentationcoefficient (corrected to infinite dilution) for anumber of the samples, is also recorded. As-suming that similarity in base composition isthe minimal requirement for extensive homologiesin base sequence between species, it was predictedthat B. natto, B. brevis, B. stearothermophilus, B.
* Per cent G+C (quanine plus cytosine) was estimated from the melting temperature of the DNA(Marmur and Doty, 1959, 1962). DNA solutions (20 lAg/ml in 0.15 M NaCl plus 0.015 M Na citrate)were heated in a Beckman DU spectrophotometer. Absorbance at 260 m,u was measured as a functionof temperature.
t B. subtilis was used as the recipient in homologous and heterologous transformation. The homolo-gous transformation was assigned the value of 100%7 for a basis of comparison. The homologous trans-formation efficiencies were 0.1 to 0.3% of the total population for the indole marker and 0.1% for thearginine marker.
I For orders of magnitude, see Table 4.
polymyxa, B. niger, B. subtilis var. niger, andB. subtilis var. aterrimus might transform B.subtilis. Since the other Bacillus species havedifferent base compositions from that of B.subtilis, the likelihood of homologous basesequences would be extremely small.
B. subtilis is transformed by DNA from B.natto and B. subtilis var. aterrimus at a higherefficiency than by homologous DNA (Table 2).The DNA samples isolated from B. niger, B.subtilis var. niger, and B. polymyxa transformB. subtilis at a low frequency. These hetero-specific transformations are 103 to 104 times lessfrequent than the homospecific transformationof B. subtilis. B. brevis and B. stearothermophilusdid not transform B. subtilis. Neither was trans-
formation, with respect to any of the threemarkers, observed with any of the DNA sampleswhich have different over-all DNA compositionsfrom that of B. subtilis.
In cases where no transformation is recorded,the results cannot be attributed to a degradationof the DNA during the isolation. The DNAsamples had a high molecular weight (about 10to 15 million, estimated from their sedimentationcoefficients), and were native, as revealed by theabsorbance-temperature denaturation profiles.The DNA samples that did transform B. subtiliswere stable, with respect to biological activityand sedimentation coefficients, over long periodsof storage at refrigerator temperatures, whenkept sterile. All the DNA samples, including
TABLE 3. Transformation of Bacillus subtiliswith respect to a heterologous indole marker
incorporated into homologous DNA
Recipient cell Transforming DNA* Transformants1Recipient_cell ml
B. subtilis B. subtilis 2.4 X 105B. subtilis-polymyxa 3.7 X 105B. subtilis-niger 5.2 X 104B. subtilis-var. niger 3.6 X 104
* The indole marker was incorporated into B.subtilis genome in a heterologous transformation(see text).
those inactive in transformation, competed withactive DNA under saturating conditions forreceptor sites of transformable cells, againindicating that those samples which did nottransform were not degraded and were capableof carrying out the initial stages of the trans-formation process.
It has been suggested (Schaeffer, 1958a, b) thatthe low frequency usually observed in hetero-transformation is due to incomplete homologyof the DNA molecules of the donor and recipientbacteria. The DNA may be homologous over agiven length of the molecule and exhibit lack ofhomology in the remainder of the molecule. Thus,poor heterotransformation could be due to eitherlack of perfect homology within the locus beingselected, or lack of homology in the remainder ofthe DNA molecule. The following experimentwas performed to test the two possibilities. B.subtilis ind- cells were transformed with DNAfrom wild-type strains of B. polymyxa, B. niger,and B. subtilis var. niger. The ind+ transformantswere selected, and DNA was isolated from them.This DNA (designated B. subtilis-polymyxa, B.subtilis-niger, and B. subtilis-var. niger, respec-tively) was used to transform the original B.subtilis ind- recipient strain. The results of thisexperiment can be seen in Table 3. If the indolelocus itself were not perfectly homologous inB. subtilis and the other species employed, theincorporation of this locus from the other speciesinto the B. subtilis genome should not affect thefrequency with which this locus can again betransmitted to B. subtilis ind- cells. If, on theother hand, the rest of the DNA moleculeslacked the microhomology necessary for perfectpairing, the "isolation" of the indole locus fromthe other species and its incorporation into the
TABLE 4. Comparison of homologous andheterologous transformation in three
B. subtilis Es B. subtilis ER 1 X 104B. subtilis var. 3.5 X 104
aterrimus ERB. natto ER 1 X 105
B. subtilis var. B. subtilis var. 730aterrimus Es aterrimus ER
B. subtilis ER 230
B. natto Es B. natto ER 50B. subtilis ER 40
* The recipient cells were sensitive to 0.1 ,ug/mlof erythromycin on solid media.
t The strains from which the DNA was isolatedwere able to grow on solid media containing 0.1,ug/ml of erythromycin. The transformants wereincubated for 90 min in the absence of erythro-mycin (expression time) and plated on mediacontaining 0.1 ,ug/ml of erythromycin.
B. subtilis genome should remove the hindranceto pairing and result in a high frequency oftransformation. The data point to lack of ho-mology in regions outside the indole locus, andto a close similarity of nucleotide sequencewithin the indole loci.Homologous and heterologous transformation
has been observed with B. natto and B. subtilisvar. aterrimus as the recipient strains (Table 4).The low frequencies of transformation observedwith these strains are most probably a reflectionof the poor ability of the cells to take up thetransforming DNA under the conditions used(Lerman and Tolmach, 1957).Another criterion for testing microhomologies
of DNA structure is the formation of DNAhybrid molecules (Schildkraut, Marmur, andDoty, 1961). When bacterial DNA is heated at100 C and annealed, renaturation of homologousstrands takes place between complementarynucleotides. Renaturation would appear torequire homologous complementary sequences ofnucleotides along the DNA strands. WhenB. subtilis DNA is heated and annealed in amixture with homologous N'5- and deuterium-labeled DNA, a hybrid molecule is formed whichcontains one strand of normal (light) and one
- - DENS TYFIG. 1. Attempt at hybrid formnation in the genus
Bacillus. B. subtilis DNA, fully labeled with N15and deuiteriumn, was heated and annealed with DNAfront B. later-osporus (A), B. sutbtilis var. aterrimus(B), and unlabeled B. szibtilis. The annealed samnpleswer-e tr-eated with Escher-ichia coli phosphodiesteraseto renmove any unrenatuired nmater-ial. The bandis (inor-der of incr-easing density) are renatured lightDANA, hybrid DNA (absent in A), and renatiiredfully labeled B. subtilis. The fully labeled B. subtilisDA7-A1 was par-tly degr-aded during the isolationprocedure and, therefore, dicd not renature a.s wellas the unlabeled DAtA sanilples.
strand of Nl5D (heavy) DNA. This hybrid DNAbands in CsCl at an intermediate density betweenthe fully-labeled and unlabeled DiNA. Thetracings of CsCl densityr-gradient centrifugationsare shown in Fig. 1. B. sulbtilis DNA forms a
hybrid band with homologous DNA and wN-ithB. subtilis var. aterrimtus DNA. No hybrid isformed, however, bet-een B. subtilis DNA andB. laterosporus DNA. The study on molecularhybrid formation is being extended to otherspecies of Bacillus.
Preliminary experiments indicate a goodcorrelation between the ability- of a DNA totransform B. subtilis and the facility with whichit forms a hybrid DNA band with N'5D-labeledB. subtilis DNA.
DISCUSSION
A number of cases of interspecific and inter-generic transformation among microorganismshave been reported. An attempt has been madeto correlate transformability with the DNAbase composition within the genus Bacillus(Marmur, Schildkraut, and Doty, 1961b) andmore recently in the genus Neisseria (Catlin,1961).
In this study, it was found that the DNAof Bacillus species that have a different basecomposition from B. subtilis are not able totransform it. It was also possible to lpredictwhich species of Bacillus might yield D)NAcapable of transforming B. subtilis. The pre-dictions held true for three unlinked geneticmarkers. Thus, of the species that have a B.subtilis-like DNA base composition, B. nattoand B. subtilis var. aterrimus transform B.subtilis with a very high efficiency. B. niger, B.subtilis var. niger, and B. polymyxa DNA trans-formed B. subtilis at a reduced frequency. B.brevis and B. stearothermophilus DNA did nottransform B. subtilis, in spite of the similarityin base composition. The above observationsindicate that the similarity- of over-all DNAbase composition is a necessary but not sufficientrequirement for the transfer and incorporationof genetic information among organisms. Noneof the other DNA samples that vary from B.subtilis DNA in their base composition couldserve as donors in transformation.
In the case of B. natto and B. subtilis v-ar.aterrim us DiNA, where a high frequenc- ofinterspecific transformation is observed, onecould speculate that a high degree of micro-homology with B. subtilis DNA exists. Thesestrains also undergo recilprocal transformationwith B. subtilis DNA. The DNA of the speciesthat give a lower level of transformation can be
thought of as having a lower order of micro-homology with B. subtilis DNA. The DNAsamples that have a similar base composition,yet do not transform B. subtilis, apparently havea different base sequence along at least one(indole-bearing) DNA molecule. It should beborne in mind that other genetic markers,residing on different DNA molecules, may revealthe existence of microhomologies not detectedin this study. In cases where partial micro-homology exists between DNA samples (asindicated by the low-frequency heterotrans-formation), one could hope to increase the extentof microhomology by partially shearing theDNA. The shearing might decrease the repulsionof noncomplementary nucleotide pairs byshortening the nonhomologous regions. Experi-ments are being undertaken in this laboratory toinvestigate the effect of shearing on heterologoustransformation and hybrid band formation.
Genetic exchange between microorganismsmight be used to study their taxonomic relation-ships. Because the biological divergencies in themicrobial world are so wide, and multiple con-tinuous variations exist in the morphological,physiological, and biochemical properties be-tween related groups, the division of bacteriainto meaningful taxonomic units has beendifficult (Stanier, 1955). Often, the criteria usedare arbitrary and in some cases lead to the classi-fication of strains into different species or genera,or both, when the differences may be due toonly a few mutational events. The use of inter-specific transformation offers an importantcriterion for taxonomic relatedness andapproaches the use of genetic compatibility inthe definition of species for higher plants andanimals. The present findings, that a closerelationship exists between B. subtilis, B. natto,B. subtilis var. niger, and B. subtilis var. aterrimususing the criterion of transformation, have beenindependently arrived at by Smith, Gordon, andClark (1952) using the more classical criteria,such as morphology and biochemical properties.
Phage-mediated transduction would alsoappear to probe the microhomologies of DNAstructure. It has been successfully used in genetictransfers between Escherichia and Shigella. Notall genetic exchanges, however, may be con-sidered indications of microhomologies. Geneticexchange by F-duction has been observedbetween Escherichia and Serratia (with dis-
similar base ratios). This exchange representsa case where the incoming genetic material doesnot become integrated into the genome of therecipient cell (Marmur et al., 1961a; Falkowet al., 1961). Genetic competence, as a test ofmicrohomology of DNA base sequence, thereforehas to be limited to exchanges that involve theintegration of new genetic material into thegenome of the recipient organism.The finding that a close relationship exists
between taxonomy, transformability, and DNAhybrid molecule formation provides a precedentfor the use of either transformation or hybridband formation as a rational approach forstudying the relatedness of microorganisms.
ACKNOWLEDGMENTS
The authors are indebted to L. Grossman forthe generous gift of E. coli phosphodiesteraseand for his helpful advice. We wish to thank C.Schildkraut for his valuable suggestions, andMrs. M. Cahoon for her expert assistance incarrying out some of the experiments.
This work was supported by grants from theU.S. Public Health Service (C-2170, RG-7985,and CRT-5033) and the National ScienceFoundation (13990).
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