HYBRIDIZATION BETWEEN ESCHERICHIA COLI AND SHIGELLA' S. E. LURIA AND JEANNE W. BURROUS Department of Bacteriology, University of Illinois, Urbana, Illinois Received for publication April 18, 1957 Genetic recombination by mating has been demonstrated among a variety of strains classified as Escherichia coli (Lederberg and Tatum, 1946; Cavalli et al., 1953). The possibility of genetic transfers among bacteria currently classified in different species or genera has been proved by transformation and transduction experiments (Lederberg and Edwards, 1953; Schaeffer and Ritz, 1955; Lennox, 1955). These findings are hardly surprising since there is no reason to expect that current bacterial classification cor- relates closely with capacity for hybridization or even with evolutionary relations (Luria, 1947). Hybridization capacity, even if present, might indeed not be responsible for significant amounts of gene flow among natural bacterial populations, which can propagate indefinitely by vegetative reproduction alone. Hence, adaptive selection might lead to considerable diversity among po- tentially interfertile clones, both because of the rarity of fertilization events and because of other isolation mechanisms. In this paper we present evidence that places the majority of dysentery bacilli (genus Shigella) into the same fertility system as Escherichia coli. The results also indicate a possible role of genetic recombination in the origin of some Shigella serotypes and, more generally, in the evolution of natural populations of these bac- teria. They suggest some potential pitfalls of current bacteriological procedures for the identi- fication of pathogenic enteric bacteria. 1 This work was supported by grants from the American Cancer Society (recommended by the Committee on Growth), from the National Insti- tutes of Health (E-1629), and from the Dazian Foundation for Medical Research. The authors wish to express their appreciation for the advice of Dr. W. H. Ewing and Dr. I. Saphra, who also generously supplied cultures and sera. The collaboration of Dr. E. S. Lennox in the initial experiments is also gratefully acknowl- edged. MATERIALS AND METHODS Cultures. E. coli strain K-12 and its derivatives were from our laboratory collection. Most of these strains were received from Drs. L. Cavalli, W. Hayes, E. M. and J. Lederberg, P. D. Skaar, and E. Wollman. E. coli strain C (Lieb et al., 1955) was also used. All the strains are motile. Shigella dysenteriae strain Sh is a rough strain, commonly used as indicator for the phages carried by E. coli strain Lisbonne-Carrere (Bertani, 1951). Type cultures of S. dysenteriae, S. flexneri, and S. boydii were received either from the Division of Laboratories, Illinois Department of Public Health, Chicago, thanks to Dr. H. J. Shaughnessy, or from the Com- municable Disease Center, U. S. Public Health Service, Atlanta, through the courtesy of Dr. W. H. Ewing. The Illinois strains are identified by the symbol I, the Atlanta strains by the symbol A (for example, S. dysenteriae strain 1I; S. flexneri strain 2aA). Streptomycin resistant mutants (Sr) were isolated from plates of solid nutrient media containing 100 ,g/ml streptomycin that had been seeded with about 1010 streptomycin sensitive (S8) cells per plate. Sr cultures were maintained and grown with 100 ,tg of streptomycin/ml, except when this would interfere with the experi- mental procedure. Phage resistant mutants were isolated by standard methods. Lysogenic derivatives were obtained from plates with con- fluent incomplete lysis. Phage sensitivity tests were done by cross smearing. Media. L broth and L agar contain: tryptone, 10 g; yeast extract, 5 g; NaCl, 10 g; glucose, 1 g; water, 1,000 ml; (pH 7.0) with or without 10 g powdered agar, respectively. Minimal, EMB and EMS (= minimal EMB) media were prepared following Lederberg (1950a) with 15 g/L agar. Sugars were dissolved in water, sterilized and added to media to a concentration of 1 per cent. Media for gas production tests were made by adding 1 per cent sugar (sterilized 461 on July 9, 2020 by guest http://jb.asm.org/ Downloaded from
Transcript
HYBRIDIZATION BETWEEN ESCHERICHIA COLI AND SHIGELLA'
S. E. LURIA AND JEANNE W. BURROUSDepartment of Bacteriology, University of Illinois, Urbana, Illinois
Received for publication April 18, 1957
Genetic recombination by mating has beendemonstrated among a variety of strains classifiedas Escherichia coli (Lederberg and Tatum, 1946;Cavalli et al., 1953). The possibility of genetictransfers among bacteria currently classified indifferent species or genera has been proved bytransformation and transduction experiments(Lederberg and Edwards, 1953; Schaeffer andRitz, 1955; Lennox, 1955). These findings arehardly surprising since there is no reason toexpect that current bacterial classification cor-relates closely with capacity for hybridizationor even with evolutionary relations (Luria, 1947).Hybridization capacity, even if present, mightindeed not be responsible for significant amountsof gene flow among natural bacterial populations,which can propagate indefinitely by vegetativereproduction alone. Hence, adaptive selectionmight lead to considerable diversity among po-tentially interfertile clones, both because of therarity of fertilization events and because of otherisolation mechanisms.
In this paper we present evidence that placesthe majority of dysentery bacilli (genus Shigella)into the same fertility system as Escherichiacoli. The results also indicate a possible role ofgenetic recombination in the origin of someShigella serotypes and, more generally, in theevolution of natural populations of these bac-teria. They suggest some potential pitfalls ofcurrent bacteriological procedures for the identi-fication of pathogenic enteric bacteria.
1 This work was supported by grants from theAmerican Cancer Society (recommended by theCommittee on Growth), from the National Insti-tutes of Health (E-1629), and from the DazianFoundation for Medical Research.The authors wish to express their appreciation
for the advice of Dr. W. H. Ewing and Dr. I.Saphra, who also generously supplied culturesand sera. The collaboration of Dr. E. S. Lennox inthe initial experiments is also gratefully acknowl-edged.
MATERIALS AND METHODS
Cultures. E. coli strain K-12 and its derivativeswere from our laboratory collection. Most ofthese strains were received from Drs. L. Cavalli,W. Hayes, E. M. and J. Lederberg, P. D. Skaar,and E. Wollman. E. coli strain C (Lieb et al.,1955) was also used. All the strains are motile.
Shigella dysenteriae strain Sh is a rough strain,commonly used as indicator for the phagescarried by E. coli strain Lisbonne-Carrere(Bertani, 1951). Type cultures of S. dysenteriae,S. flexneri, and S. boydii were received eitherfrom the Division of Laboratories, IllinoisDepartment of Public Health, Chicago, thanksto Dr. H. J. Shaughnessy, or from the Com-municable Disease Center, U. S. Public HealthService, Atlanta, through the courtesy of Dr.W. H. Ewing. The Illinois strains are identifiedby the symbol I, the Atlanta strains by thesymbol A (for example, S. dysenteriae strain 1I;S. flexneri strain 2aA).
Streptomycin resistant mutants (Sr) wereisolated from plates of solid nutrient mediacontaining 100 ,g/ml streptomycin that had beenseeded with about 1010 streptomycin sensitive(S8) cells per plate. Sr cultures were maintainedand grown with 100 ,tg of streptomycin/ml,except when this would interfere with the experi-mental procedure. Phage resistant mutantswere isolated by standard methods. Lysogenicderivatives were obtained from plates with con-fluent incomplete lysis. Phage sensitivity testswere done by cross smearing.
Media. L broth and L agar contain: tryptone,10 g; yeast extract, 5 g; NaCl, 10 g; glucose,1 g; water, 1,000 ml; (pH 7.0) with or without10 g powdered agar, respectively. Minimal,EMB and EMS (= minimal EMB) media wereprepared following Lederberg (1950a) with 15g/L agar. Sugars were dissolved in water,sterilized and added to media to a concentrationof 1 per cent. Media for gas production testswere made by adding 1 per cent sugar (sterilized
separately) to tubes of nutrient broth (pH 7.2)with bromthymol blue indicator and invertedvials. Assays of phage X were done on the mediumrecommended by Kaiser (1955). Phage P1 was
assayed on L agar plus 10-2 M CaC12-Antisera. Sera against Shigella type cultures
were obtained either commercially (MarkhamLaboratories, Inc.) or through the courtesy ofDr. W. H. Ewing and Dr. I. Saphra. Sera (anti-0)against E. coli strain K-12, its derivatives, andhybrid strains were prepared in rabbits by 1 or
2 injections of about 109 cells collected fromnutrient agar, heated at 100 C for 2.5 hr andmixed with adjuvants (Freund's method, see
Cohn, 1952). Adsorbed sera were prepared bythe double adsorption method according toEdwards and Ewing (1955) using cells from 10plates of L agar/ml of serum.
Agglutination tests were done in 9 mm tubes,using 0.5 ml vol of saline. The tubes were keptat 56 C for 2 hr, then at room temperature over-
night. The antigens consisted of cells collectedfrom agar slants or from heavy broth cultures,with or without washing. Whether used alive,or heated for 1 hr at 100 C, or treated with 75per cent ethyl alcohol or with 0.5 per centformalin overnight, the cells of a given organismgave 0-agglutination endpoints agreeing within a
factor of 2. In most tests, formolized antigenswere used for nonmotile cultures and alcoholtreated antigens for motile ones. A rapid diag-nostic test for serological changes in hybrids willbe described in a later section.
RESULTS
Selection of recombinants. Strains to be crossedmust exhibit stable differences in characters so
that recombinants can be enriched and detectedselectively. A convenient method proved to beselection for lactose utilization in streptomycincontaining media (Lederberg, 1951; Hayes,1953b). Recombinants Lac+Sr can be obtainedfrom mixtures of Sr Shigellas and Ss (strepto-mycin sensitive) E. coli parents plated on EMBlactose agar with 100 to 200,g/ml streptomycin.All Shigella strains proved stably Lac-; platedalone, they gave no Lac+ papillae on EMB-lactose agar and no detectable growth in minimalmedia with lactose as only carbon source (inaddition to necessary growth factors); nor was
any ,B-galactosidase detectable in these strainsby the o-nitrophenyl-,3-D-galactoside test (Leder-
berg, 1950b). Sr cells appear with a frequencylower than 10-9 in SI cultures of E. coli strainK-12 derivatives.
Sr derivatives of the Shigella strains to betested were isolated first. Heavy suspensionsfrom stationary cultures of these Shigellas(Lac-Sr) and of E. coli (Lac+SS), grown in Lbroth to about 108 cells/ml, chilled and collectedby cold centrifugation, were mixed directly onEMB-lactose streptomycin agar. Lac+ papillae,when present, appeared in 2 to 3 days. Thesepapillae, upon isolation by streaking on EMBlactose, yielded stable Lac+Sr clones, whoserecombinant nature was confirmed by tests forother characters in which the parent strainsdiffered (unselected markers, see below). TheLac+ recombinants gave a positive test for,-galactosidase. They produced acid from lactosein 18 to 24 hr (pH 4.9 i4 0.2) and no gas.The parental combinations that were tested
by this method are shown in table 1. The parentalShigella strains never yielded a single Lac+mutant in any of the control plates, which con-tained altogether over 1011 cells. Note that Lac+recombinants were obtained also in crosses ofShigella with E. coli strain C, a strain whichdiffers from K-12 in many respects (Lieb et al.,1955).Frequency of recombination and polarity. The
frequency of recombinants Lac+Sr (or Lac+prototrophs, see below) as a function of thepolarity (Hayes, 1953a; Cavalli et al., 1953) ofthe E. coli parents is shown in table 2. In theseand in all other crosses, the Shigella parents be-have as typical F- strains. All Hfr cultures ofE. coli (see Hayes, 1953b) give higher frequenciesof recombination than F+ cultures with Shigellasas well as with other strains of E. coli. Thus,the mating polarity system observed in E. coliextends to Shigellas as well. The frequencies ofrecombinants are not much higher when theparents are incubated together before platingthan when they are mixed on plates. The fre-quencies are uniformly lower for crosses of F+ orHfr E. coli with Shigellas than with F- E. coli.It will be shown below that this difference is dueto lower effectiveness of matings in producingrecombination rather than to lower frequency ofmating.
Unselected markers and linkage. Recombinantsisolated from crosses are expected to containvarious assortments of the genetic traits, other
TABLE 1Fertile combinations of strains as tested by the Lac+Sr selection
Samples of 0.1 ml from each culture were mixed directly on EMB-lactose-streptomycin agar. Lac+colonies were isolated by restreaking on the same medium.
* I = strains from the Division of Laboratories, Illinois Department of Public Health, Chicago,Illinois.
t A = strains from the Communicable Disease Center, U.S.P.H.S., Atlanta, Georgia.
than the selected ones, in which the parentstrains differed ("unselected markers"). Gen-erally, more characters are contributed to thehybrids by the F- parent than by the F+ or Hfrparent (Hayes, 1953b). The relative frequenciesof various unselected markers and their depend-ence on the choice of selective markers make itpossible to map the corresponding genetic factorsin a linear linkage map (Lederberg, 1947).
In the present work we have not attempted toconstruct a detailed genetic map of any one
strain of Shigella. A number of characters were
used either as selective or as unselected markers,mainly in crosses between E. coli strain K-12,S. dysenteriae strain Sh and S. flexneri strain2aI and their derivatives. The characters thatwere tested for presence or absence in hybrids
are listed in table 3. As expected, the hybridsare predominantly like the Shigella F- parents.Note that some characters from the F+ or Hfrcoli parents never appeared in the hybrids. Thisis true for production of indole, for resistance toazide, and for utilization of xylose, maltose andmannitol with S. dysenteriae strain Sh; for mo-
tility and gas production both with strain Shand with S. flexneri strain 2aI.Any character that appears in hybrids as a
nonselected marker can also be used as a selectivemarker. For example, selection was successfulfor Ara+Sr hybrids in crosses of S. dysenteriaestrain Sh X E. coli, and for Xyl+Sr (or Dul+Sr,or Rha+Sr) with S. flexneri strain 2aI. All Ara+and Xyl+ recombinants produce acid in 24 hr
laI X E. coli2aI X E. coli2aI X E. coli2aA X E. coli2aA X E. coli2bA X E. coli2bA X E. coli4aI X E. coli4aA X E. coli
TABLE 2Fr equency of recombination and polarity of the Escherichia coli parent
Examples of recombination frequencies obtained in crossing Shigella cultures with derivatives ofE. coli strain K-12.
RatioExperiment Shigella and Escherichia coli Parents: Organism, Strain Selection for Recombinants/
Hf r or F+ cells
I* S. dysenteriae 57-1 prototroph X E. coli F-M- Lac+M+ <10- (none)1-31-55 S. dysenteriae 57-1 prototroph X E. coli F+C-H- Lac+C+H+ 10-7
S. dysenteriae 57-1 prototroph X E. coli Hfr M-Ss Lac+M+ 2 X 10-5S. dysenteriae 57-1 prototroph X E. coli Hfr M-Sr Lac+M+ 4 X 10-5
lit S. dysenteriae Sh Sr X E. coli F+(X) prototroph Ss Lac+Sr 1.4 X 10-71-8-57 S. flexneri 2aI Sr (X) X E. coli F+(X) prototroph Ss Lac'Sr 6.6 X 10-7
E. coli F-T-L-Sr (X) X E. coli F+(X) prototroph 5s T+L+Sr 1 X 10-5
IlIt S. dysenteriae Sh Sr X E. coli Hfr S" Lac+Sr 6 X 10-412-13-56 S. ftexneri 2aI Sr X E. coli Hfr S" Lac+Sr 3 X 10-4
E. coli F-T-L-Sr X E. coli Hfr Ss T+L+Sr 7 X 10-1
* Cultures mixed directly on plates of EMS-lactose-agar.t Mixtures containing 3-12 X 101 F- and 3.0 X 101 F+ cells; incubated for 40 min with aeration be-
fore plating. Assays after 30 min of aeration.$ Mixtures containing 5-8 X 109 F- cells and 7 X 106 Hfr cells; incubated for 40 min in broth without
aeration before plating. In this experiment the number of Hfr cells was measured at the time of mixing.Platings in experiments II and III were done on EMB-lactose-streptomycin-agar for the Shigella X
E. coli crosses; on minimal-thiamine-streptomycin-agar for the E. coli X E. coli crosses. The mediumused in the Shigella X E. coli crosses may allow additional recombination on the plates.
Symbols: T = threonine; L = leucine; M = methionine; C = cysteine; H = histidine; S = strep-tomycin.
TABLE 3Transmission of characters from Escherichia coli parents to hybrids
Code: Lac = lactose. Ara = arabinose. Xyl = xylose. Rha= rhamnose. Dul = dulcitol. Mal =
maltose. Mtl = mannitol. Ind = indole production. mot = motility. aer = gas production. Az =
azide. S = streptomycin. Nic = nicotinamide. Glt = glutamate. V1_5 = phages Ti, T5. V6 = phageT6. P1 = Phage P1. X-C = phage X produced like that from E. coli strain C. X-K = phage X pro-duced like that from E. coli strain K-12. Lacr- = cryptic. $-D-galactosidase. + = utilized; or syn-thesized; or lysogenic; or present. - = not utilized; not synthesized; not lysogenic; or absent. 8 = sen-sitive. r = resistant.
(and no gas) in liquid media with the correspond-ing sugars.
S. flexneri strain 2aI grows in minimal mediumsupplemented with nicotinamide and glutamate(Nic-Glt-). In crosses of S. flexneri strain 2aIwith auxotrophic F+ or Hfr strains of E. coli(M- or T-L- or C-H-) no prototrophic re-combinants were observed. Crosses on minimalagar + nicotinamide + glutamate + lactoseyielded only Lac+ Nic- Glt- recombinants. Thus,there was no evidence for transfer of Nic+ andGlt+ from E. coli to hybrids.
S. dysenteriae strain Sh has ill-defined, non-specific requirements for a variety of amino acids,but it readily yields variants that grow fairlywell on mninimal medium. One such variant,strain 57-1, obtained from a Sr derivative ofstrain Sh, has been used extensively in our work.In crosses of Shigella 57-1 X E. coli, we canselect hybrids either for Lac+Sr or for Lac+prototrophy (on EMS minimal agar with lactoseas only carbon source) as shown in table 2. Thelatter selection allows the isolation of recom-binants with the S' character from an E. coliparent.The frequency of unselected markers permits
us to establish a few linkage relations. Crossesof Shigella Sh (or 57-1) X E. coli suggest thefollowing linkage pattern: Ara-V1, 5-Lac-V6(or possibly Ara-V1, s-V6--Lac). Here Araand Lac represent the loci whose alleles arepresent in the Shigella parent. In crosses withS. flexneri, Lac is closely linked to V1, 5 and onlydistantly linked to Rha and Xyl. The linkagerelation between the loci V1, 5, Lac, V6 andAra is similar in Shigella and in E. coli strainK-12 (Cavalli-Sforza and Jinks, 1956). This in-dicates extensive homology of genetic organiza-tion.
It is impossible to decide, on the basis of ourpresent data, whether the characters of the F+(or Hfr) coli parents that fail to appear in hy-brids are excluded by the choice of selectivemarkers (linkage with markers against whichselection was made), or by incomplete homologybetween the linkage systems of E. coli and Shi-gella, or by chromosomal rearrangements. Somecharacters, such as motility and aerogenesis,may involve multigene control. Failure of ex-pression of a gene may be due to interactionwith other genetic factors. These problems may
be resolved more easily by use of transduction(Lennox, 1955).An interesting cross, included in table 3, is
one in which the E. coli parent carries the muta-tion Lac1- (cryptic 3-galactosidase) (Lederberget al., 1951). Adaptive synthesis of f-galactosidasecan be induced more readily by alkyl-3-galacto-sides than by lactose. Crosses on EMB-lactose-streptomycin agar yield late papillae, from whichone can isolate bacteria that form ,-galactosidasewhen exposed to methyl-,B-galactoside. Thus,the hybrid has acquired the same Lacg- allelepresent in the E. coli parent.
Behavior towards phages PI and X. Strains ofE. coli and of Shigella are hosts for many commonphages (including the T1-T7 phages), with in-dividual differences in host range. A special inter-est applies to phages P1 and X, the former as anagent of transduction (Lennox, 1955), the secondas a phage whose prophage location in the linkagemap of E. coli strain K-12 and whose behaviorin bacterial crosses have been investigated(Wollman et al., 1956).The host range of these phages among Shigella
cultures is shown in table 4. The widespread sensi-tivity to phage P1 opens the possibility of geneticanalysis by means of transduction. Note alsothat some Shigella strains, like S. dysenteriaestrain Sh, are susceptible to P1 with its originalhost range as first isolated from E. coli strainLisbonne-Carrere (Bertani, 1951); other strains,like S. flexneri strain 2aI, are only lysed by themutant Plk, isolated by its ability to lyse E.coli strain K-12. This observation suggests fur-ther mutational homologies between Shigellasand E. coli.Phage X attacks only S. flexneri strain 2aI;
the other Shigellas are neither lysed nor lysogenicfor X. S. flexneri strain 2aI is readily lysogenized.When X is liberated by this strain, either byinduction of a lysogenic culture or by infectionof a sensitive culture, it is modified in the sameway as when grown on E. coli C (Bertani andWeigle, 1953); this X-C phage fails to grow inmost cells of (nonlysogenic) E. coli strain K-12derivatives. This indicates one additional genetichomology between a Shigella and E. coli strain C.Several hybrids from strain 2aI X E. coli strainK-12 were tested and all retained the C-likemodifying property.
TABLE 4Sensitivity of various strains to phages P1 and X
Phage P1 Phage XOrganism - ______ Kind of Phage X
Wildtype k Mutant, Grown on Grown on PoueOrgmWild type active on K-12 E. coli K-12 E. coli C
Escherichia coli K-12............ R S S s X-KE. coli C ........................ S S S S X-CShigella dysenteriae Sh .......... S S R RS. dysenteriae 1A ................ R RS. dysenteriae 2A ................ R R R RS. dysenteriae 5A ................ R R R RS. dysenteriae 6A ................ R R R RS. dysenteriae 7A ................ R R R RS. dysenteriae 8A ................ R S R RS. flexneri 2aI and hybrids ....... R S S S X-CS. flexneri 2aA and hybrids .S S R RS. flexneri 2bA .................. R S R RS. flexneri 4aA................... S S R RS. boydii 4A..................... R R R RS. boydii 6A..................... S S R R
S = sensitive; R = resistant by spot test; s = sensitive with low efficiency ofstrains from Atlanta; I = type strains from Chicago.
crosses of S. flexneri strain 2aI with X lysogenicE. coli strain K-12 in order to avoid "zygoticinduction" (Jacob and Wollman, 1956), that is,maturation of X and lysis of the F- parent whenprophage X is transferred from a lysogenic F+or Hfr mate.The phenomenon of zygotic induction of X
made it possible (Wollman et al., 1956) to esti-mate the frequency of mating, as distinct fromthe frequency of recombination, which dependson the frequencies of mating, of transfer of theselective markers from the F+ or Hfr parent,and of postzygotic integration of these markersinto the genome of the hybrids. Experiments onzygotic induction were done with E. coli cultureHfr H B1- (X), a strain that was used extensivelyby Wollman et al. (1956) in their analysis of themechanism of recombination, details of whichneed not be given here. We need only recall thatthe order of gene transfer from this Hfr strainto a coli F- mate is: T-L-Lac Gal-X. Underoptimal conditions, mating can involve nearly100 per cent of the Hfr cells. Zygotes that receiveX are lysed; when mating is allowed to go tocompletion, this lysis eliminates about 50 percent of the zygotes. This system made it possibleto compare zygotic induction and recombinationusing either Shigellas or E. coli as the F-. Thesame Hfr strain, but nonlysogenic, was used to
plating; A = type
estimate recombination frequencies undisturbedby zygotic induction of X (table 5). In the re-combination experiments selection was made forLac+Sr when using Shigellas F- and for (TL)+Srwhen using E. coli F-.The results in table 5 show that, on the one
hand, zygotic induction (and therefore matingand gene transfer) is about equally frequent inall cases. On the other hand, the recombinationfrequency is 100 to 1000 times lower in crosseswith F- Shigellas than with F- E. coli. Thisis presumably not due to the different selec-tive markers since, at least in crosses withthe F- E. coli, the Lac locus is donated to re-combinants about 50 per cent as often as the TLsegment (Wollman et al., 1956). We concludethat the lower recombination reflects lower post-zygotic integration of genes from the Hfr parent,rather than less frequent mating or less frequenttransfer of genetic material. Test for a possibledeath of Shigella F- cells following mating withE. coli Hfr gave negative results.A remarkable observation is that the phage
X liberated upon zygotic induction in the matingof Hfr (X) with S. flexneri strain 2aI is onlypartly (-50 per cent) modified to the X C form,although phage grown directly on strain 2aI isfully modified. In control experiments withE. coli strain C F- the phage produced by zygotic
induction was more extensively modified to theX * C form, although not as completely as reportedby Jacob and Wollman (1956). Experiments arein progress to clarify the reason for the partialmodification. It seems possible that a relationexists between the low level of postzygotic in-tegration and the persistence of the X K formin coli X Shigella matings.Note that zygotic induction occurs also in
crosses with S. dysenteriae strain Sh, which can-not absorb phage X and which, apparently, doesnot become X-lysogenic upon mating. Growth ofX transduced by phage P1 into S. dysenteriaestrain Sh has also been observed by Lennox (to bepublished). Zygotic induction of X was absent,however, in a cross with S. flexneri strain 2aAF-.
Unstable hybrid strains. Most hybrid strains arestable for all selected and unselected parentalmarkers. Moreover, each recombinant colonyappears to be pure (nonsegregating) for allmarkers. The one exception concerns some crossesof S. flexneri strain 2aI and its derivatives withone strain of E. coli (Hfr M-V6r Vy15r). Thesecrosses yield a certain proportion of unstableLac+ colonies, which segregate out stable Lac-,unstable Lac+ and a few stable Lac+. The Lac+unstable hybrids are stable for other charactersderived from the Hfr parent (except sometimesfor V1,5, which is closely linked to Lac). It seemslikely that in mutating from V1,5" to V1,5R theparent Hfr strain has acquired a factor resemblingthe Het factor described by Lederberg (1949).Further study of this factor is planned. Thestable Lac+ segregants resemble other Lac+recombinants from S. flexneri strain 2al, exceptin a serological property to be discussed below.
Changes in mating polarity. Most F- strains ofE. coli can be converted to F+ by infection uponcultivation in admixture with F+ strain (Cavalliet al., 1953; Hayes, 1953a; de Haan, 1954). Thisinfection may be the expression of an incompletemating (Wollman et al., 1956). The conversion ofShigellas to the F+ state was accomplished anddemonstrated by experiments of the type shownin table 6. Each of 3 Shigella strains was culti-vated in mixture with an F+ strain of E. coliand reisolated at intervals by selective platingmethods. Several of these isolates (and theoriginal Shigella strains) were then subculturedwith an F- strain of E. coli (TLB1)-. The colistrain was in turn reisolated and tested for fer-tility with E. coli ThM-. A positive test indicates
that the Shigella strain used had become F+ anda source of F+ contagion. The results were uni-formly negative for the original Shigellas andpositive for the treated ones, confirming the F-nature of the former and their conversion to F+by infection.
Repeated attempts, however, to demonstraterecombination between these F+ Shigellas andany F- strain, either E. coli or Shigella, using alimited variety of selective methods, gave onlynegative results. Whether this is due to limita-tions of our tests or to an intrinsic property ofF+ Shigellas cannot be decided from the avail-able data.
Serological properties of hybrids. In aggluti-nation tests for 0-antigens, E. coli strain K-12and its derivatives give only some slight crossreactions with the Shigella strains tested in ourwork.2 When certain hybrids were tested withunadsorbed 0-antisera against the parent strains,a new character appeared. Most Lac+ hybridsfrom crosses between E. coli strain K-12 de-rivatives and S. flexneri strain 2aI were found togive a reduced agglutinin titer with a com-mercial antiflexner "W" serum. This findingprompted further serological investigation ofthese and other hybrids.Most of the Lac+ hybrids from crosses between
E. coli (whether derivatives of strain K-12 or ofstrain C) and S. flexneri strain 2aI show thealtered serological behavior with antiflexner sera,which consists in a reduced agglutinating titerwith unadsorbed sera antiflexner W and anti-flexner 2a, with increased agglutinability byseveral sera antiflexner 4a. These results areshown in table 7. Sera against other flexnerprototypes were not tested.Two Lac+ hybrids with the abnormal behavior,
labeled strains F21 and F22, one X-lysogenic, theother not, were selected and used to prepareantisera. These sera prove identical in range ofreactivity. As shown in table 8 for the serumanti-F22, these sera gave high titers with thehomologous hybrid strains and low titers with
2 Most cultures of Escherichia coli strain K-12and its derivatives rapidly become rough underlaboratory conditions. The sera were preparedwith smooth cultures, which did not give self-agglutination in isotonic saline after overnighttreatment with 70 per cent ethanol. In mostagglutination tests the antigens were suspensionsprepared from smooth colonies of an Hfr M-derivative.
TABLE 6Transfer of F+ property between Escherichia coli and Shigella
Crosses (on Minimal Agar + Thiamin) Recombinants
E. coli F-(TLB,)- X E. coli F-(MB)-E. coli+ F-(TLB1)- X E. coli F-(MB)- +E. coliii F-(TLB1)- X E. coli F-(MB)- _E. colii+ F-(TLB,)- X E. coli F-(MB)- +E. coliiil F-(TLB,)- X E. coli F-(MB)- _E. coliiii+ F-(TLB1)- X E. coli F-(MB)- +E. ColiII1+ F-(TLBl)- X E. coli F-(MB)- +
Initial cultures: I = S. dysenteriae 57-1 Sr; II = S. flexneri 2a1 Sr; III = S. flexneri 2aA Sr.Mixed cultivation with E. coli F+(TLB1)-S". Inoculum: 106 E. coli + 105 Shigella. Two or three daily
transfers of 0.1 ml into 50 ml broth.Reisolation of Shigellas I, II, III (F+?) on streptomycin agar; labeled I+, II+, III+.Mixed cultivation of Shigellas I, II, III and I+, II+, III+ with E. coli F-(TLB1)-. Inoculum: 106
Shigella + 104 E. coli. Two daily transfers as above.Reisolation of E. coli by washing and streaking on minimal agar plus thiamin. Labeled E. colij,
E. colii+ . . . .
TABLE 7Agglutination reactions of Shigellaflexneri strain 2aI and of hybrids
cultures of S. flexneri strains 2aI, 2aA and 2bA.Instead, the antihybrid serum agglutinatescultures of S. flexneri type 4a as much as its ownhomologous antigen. It agglutinates E. colistrain K-12 only with very low titer.
All Lac+ hybrids from S. flexneri strain 2aIthat were tested gave either the typical "F22-like" reactions shown in table 8 or normal 2a re-actions, without intermediates. It was possible,therefore, to devise a simple test for extensive
screening of hybrids. This consists in inoculatingeach hybrid (from a pure restreaked colony) into2 tubes with 1 ml of broth each, one containingserum anti-W diluted 1:3200, the other withserum anti-F22 1:800. Upon growth overnight at37 C each hybrid is agglutinated in one or theother tube, but never in both. Several crosses be-tween cultures of S. flexneri strain 2aI and deriva-tives of E. coli strain K-12 gave 95 per cent F22-like Lac+ hybrids and 5 per cent serologically un-
changed Lac+ hybrids. Out of 100 hybrids selectedfor markers other than Lac+, the only two thatwere F22-like serologically were also Lac+.
Further crosses showed that most Lac+ hybridsbetween E. coli and another strain of S. flexneritype 2a (= 2aA) were serologically similar(although not identical) to the F22-like hybrids.That is, they had lost some titer with anti-2aand often also with anti-2b sera, and had gen-
erally become reactive at higher titer with serum
anti-F22 and serum anti-4a. An unexpectedfinding was that very similar hybrid strains were
obtained by crossing E. coli with S. flexneritype 2b. Also, hybrids with similar propertieswere obtained from crosses of E. coli with S.flexneri type 4a. The data are included intable 8.These results suggested that all changed hy-
brids belonged to a single serological group. Toassess the meaning of this hybrid group, we con-
sidered its serological relationship to E. colistrain K-12, to S. flexneri type 4a and to S.flexneri type Y. Tests on hybrids F21 and F22,kindly carried out by Dr. W. H. Ewing, classifiedthese strains as S. flexneri type Y.We tested all the hybrids, the parent cultures,
and a culture of S. flexneri type Y with serum
against E. coli K-12 and also with a number ofanti-flexner sera that had been cross-adsorbed invarious ways. The results, shown in tables 8 and9, can be summarized as follows:
(1) The Y culture gives reactions similar tothose given by the hybrids, although it is gen-
erally more reactive than any of them.(2) E. coli strain K-12 cross-reacts to a limited
but significant extent with S. flexneri type 4a;less with other type Shigellas. The hybrids vary
somewhat in their reactions with anti-K-12serum, but generally react with it less thancultures of flexner 4a.
(3) The agglutinins of antihybrid F22 serum
cannot be adsorbed completely by the parent
strains of S. flexneri type 2a nor by 2b, but arecompletely removed by adsorption with any oneof several 4a strains and by a strain of S. flexneriY.
(4) Adsorption of sera anti-2a or anti-2b withany one of the hybrids, as well as with the Ystrain, removes heterologous reactions to thesame extent.
(5) Adsorption of several anti-4a sera with anyof the hybrids, including those derived from S.flexneri type 4a itself, as well as with Y, removesall agglutinins for 2a, 2b, Y and all the hybrids,leaving various amounts of homologous ag-glutinins, depending on the serum. Results withone serum are shown in tables 8 and 9. Otheranti-4a sera are even more radically adsorbed bythe Y and hybrid strains.We conclude that the various hybrids, al-
though probably not identical to one another,form a closely related group, similar to S.flexneri type Y, relatively closer to S. flexneritype 4a than to 2a or 2b, and whose antigenicstructure includes the "group antigens" com-mon to 4a, 2a and 2b. Note that such Y-likehybrid strains are obtained also from S. flexneritype 2b, which, according to its known anti-genic composition, should, by loss of the typeantigen, give rise to X-like variants, not to Y-likeones (Edwards and Ewing, 1955). This obser-vation, and the fact that the agglutinins in anti-hybrid serum are only partially removed by ad-sorption with the parent S. flexneri type 2a(or 2b) prove that the serological change broughtabout by hybridization is not merely a loss oftype antigen of the Shigella parent. The sig-nificance of these changes as to the possible originand relations of various Shigella and E. coliserotypes will be discussed later.The following points may be emphasized.
First, the serological changes to the hybrid typeare not due to the addition or substitution of amajor E. coli antigen for the Shigella antigens inthe hybrids, since cross reactions of the hybridswith E. coli strain K-12 are only slight. Ap-parently, the genetic factor(s) responsible for thechange does not reach full phenotypic expressionin E. coli, presumably because of the differentgenetic background. (Serological tests on E. colistrain C were prevented by its roughness.)
Second, the genetic factor(s) responsible forthe change must be closely linked to the Laclocus, but are not located at the Lac locus, nor
are they another expression of the Lac+ property.In fact, about 5 per cent of the Lac+ recombin-ants are serologically unchanged. It is notable,although unexplained, that all stable Lac+derivatives from the unstable Lac+ hybrids be-long in the serologically unchanged group.The linkage of the antigen-controlling factor to
the Lac locus, used as a selective marker in ourcrosses, appears therefore to be a fortuitouscoincidence. No other determinant of serologicalspecificity has been detected yet in our ratherlimited range of crosses, using only a few strainsof Shigella, a few selective markers, and rathercrude serological tests. Further exploration is inorder.
Attempted crosses Salmonella X E. coli. Theexistence of coliform, Lac+ cultures whichferment lactose in 24 hr, have antigens identicalto those of Salmonella newington (group E2,antigenic formula 3,15: e,h-1, 6) and give riseto Lac- variants (Seligmann and Saphra, 1946),suggested their possible origin as hybrids with E.coli. It seemed desirable to attempt crossing E.coli cultures F+ or Hfr with some Salmonellasbelonging to group E2. Cultures of S. cambridge,S. newington, S. new brunswick, S. kinshase,S. selandia, and of a Lac- variant of culture# 3534 (Saphra and Seligmann, 1947) were madeSr and plated on EMB lactose streptomycinagar with strains of E. coli Lac+ S", either F+or Hfr. The results were uniformly negative.3
DISCUSSION
Many bacteria included in the genera Escher-ichia and Shigella have enough characteristics incommon to suggest a close evolutionary relation-ship. They share, for example, susceptibility tocertain phages and a common over-all pattern ofcatabolic reactions (although the fine details ofcatabolism and biosynthesis in Shigella remainlargely unexplored). The main physiological dif-ferences, which are useful for practical purposes ofclassification because of stability and good cor-relation with presumed presence or absence ofpathogenicity, include characters such as abilityto utilize lactose. Yet, as stable as these characters
3 Equally negative results were obtained in aattempt to obtain recombinants between Esch-erichia coli and a strain of Pasteurella pestis,kindly supplied by Dr. T. W. Burrows, who hadobserved that this strain and E. coli share sus-ceptibility to several phages.
are in many strains, they are known to be con-trolled by genetic factors that are mutable inother strains. In the absence of hybridizationtests, well established stable organisms can bestbe considered as "normotypes" for purposes ofclassification.Our experiments have proved that Shigella can
mate with E. coli and shares with it a commonsystem of mating polarities. By hybridization wecan create hybrids that would be considered asmonstrosities from the standpoint of traditionalbacterial classification, such as strains of S.dysenteriae that promptly ferment lactose (orarabinose or both), of S. flexneri that fermentlactose (and xylose or rhamnose) and so on. Oncethe already suspected existence of extensivegenetic homology between coli and dysenterybacilli has been confirmed by hybridization, thesefindings are not at all surprising. Indeed, itshould be possible, and might be desirable forpractical reasons, to decide by genetic analysisthe reason for the apparent stability of char-acters such as Lac- in most strains of Shigella.Such stability may reflect intrinsic gene proper-ties, or, more likely, the presence of multiplegenetic blocks, or the absence of genetic loci(chromosomal deficiencies).The failure of certain characters of E. coli
parents to appear in hybrids suggests that thegenetic homology is incomplete, and this may alsobe reflected in poor chromosomal pairing. Suchpoor pairing could underlie the low frequency ofintegration of E. coli genes into Shigella, in spiteof the high frequency of mating revealed byzygotic induction. Similar observations have beenreported in transformation experiments withHaemophilus; the frequency of integration ofnewly introduced genetic determinants is lower,the more distant the relationship between donorand recipient strains (Schaeffer, 1956). These ob-servations have also been inferpreted as reflectinginadequate pairing between genetic structures.The availability of two methods of genetic
recombination between E. coli and Shigella,mating and transduction by phage P1 (Lennox,1955), makes it certain that the taxonomicstructure of this group could be placed on a soundgenetic basis. In the present state of bacterialgenetics, however, when the nature of the matingprocess has just begun to be clarified (Wollmanet al., 1956) and the genetic structure of only oneor two strains of E. coli is known in some detail, a
detailed genetic analysis of any one Shigellawould not seem too profitable. The establishmentof a chromosomal basis for the evolution andtaxonomy of this group of bacteria is a distanthope, although a definitely realizable one.We now know that strains of Shigella and
Escherichia are potentially interfertile organisms.Therefore, three main questions arise:
(1) How widespread is interfertility amongother bacteria, at least among the Enterobac-teriaceae?
(2) Does hybridization occur in nature, and ifso, what role does the resulting gene flow play inthe variability, survival, and evolution of theseorganisms?
(3) What role, if any, does hybridization play increating the range of practically important nor-motypes of Shigella that occur in nature?The answer to the first question, of the extent
of potential interfertility among the bacterialgroups, can only be guessed. Our few attempts tohybridize Salmonella and E. coli have failed, andno positive results seem to have been reportedfrom elsewhere. The criterion of cross-sensitivityto phages, which probably reveals genetichomology in view of the relations between pro-phages and bacterial chromosomes (Stocker,1955), is of limited use since few phages affectbacteria belonging to different "genera" ofEnterobacteriaceae (except for Escherichia, Shi-gella, and related groups). The existence of un-usual common properties (especially antigens)among strains classified in different groups maybe a good guide to choice of cultures for furtherstudies. These should take into account thepossible existence of still unrecognized matingsystems, with unsuspected polarities and re-strictions in hybridizing capacity.The question of the possible occurrence in
nature of hybridization between Shigella andEscherichia, and possibly other groups, recallssuggestive observations by a number of workers.All experts in the diagnosis of enteric pathogens(Kauffmann, 1954; Weil and Saphra, 1953;Edwards and Ewing, 1955) have encounteredcultures that appear to share properties of 2 (ormore) well established normotypes and whichcould easily be explained by postulating a hybridorigin. To choose but a few examples, we maypoint first to entire groups of bacteria that fallinto these "hybrid" classes: the Arizona group,which includes Suc+, Lac+ organisms that might
be recombinants between Salmonella and E. coli;the Alkalescens-Dispar group, nonaerogenic,nonmotile organisms, including Lac+ ones, whichmight be hybrids between E. coli and S. flexneri.Also some cultures (Seligmann and Saphra, 1946;Saphra and Seligmann, 1947) ferment lactose aspromptly as E. coli but are antigenically identicalto known Salmonella strains and can, by a singlemutation Lac+ --+ Lac-, become indistinguishablefrom the latter organisms. Likewise, organismsof the Alkalescens-Dispar group are antigenicallyrelated to certain S. flexneri normotypes on theone hand, to typical E. coli strains on the otherhand.
Individual strains with 0-antigens in commonwith known coli, flexneri and Salmonella cultureshave been described (Bernstein et al., 1941;Saphra and Wassermann, 1945). Coliform or-ganisms related to Shigellas (Ewing, 1953) haveraised important questions concerning patho-genicity and diagnosis (Stuart et al., 1943). Theexperts have been unanimous in emphasizing theexistence and importance of "intermediate" or-ganisms inbetween the major normotypes (Ed-wards and Ewing, 1955). So long as vegetativereproduction was thought to be the only re-productive process in bacteria, most authorsconcerned with bacterial evolution have inter-preted these intermediate organisms as variantsfulfilling or recapitulating a mutational history ofthe Enterobacteriaceae. Yet, the possibility of ahybrid origin has not escaped some workers. Forexample, Saphra and Wassermann (1945)state: ". . . originally highly different forms mighthave a tendency of acquiring more and moresimilarity of antigenic properties. One mightdesignate such a working hypothesis as a 'hy-pothesis of convergent development' in contrastto the 'hypothesis of divergent development' ofWhite. Influences connected with the adjustmentof life in the intestinal tract and possibly the in-fluence of one species upon another during theirco-existence in the intestinal tract might favorsuch a development. The possibility that onespecies might impress its antigen upon anotherhas been experimentally proven in the classicaltransformation experiments on pneumococci byGriffith and by Dawson."
Indeed, the normal habitat of Enterobacteria-ceae, that is, the intestine of mammals and otheranimals, is quite conducive to hybridization,because of the presence of ubiquitous E. coli
strains in populations of enormous size and of thechances for numerous collisions (and other in-teractions, such as phage transfer) among cells ofthese strains and of any newcomer organism.Our experiments establish the reality of thegenetic interaction and justify the search for itsoccurrence in nature and for the role that theresulting gene flow may play in bacterial evolu-tion. This task has more than purely biological orgenetic interest. It may hold the key to thewhole epidemiology of enteric diseases; it addsmany potential dimensions to the tremendouslyimportant work of tracing and identifying theenteric pathogens.
Thus, for example, all our Lac+ hybrids, al-though they are predominantly "Shigellas"(and probably still pathogenic) would never bedetected as potential pathogens by the routinediagnostic procedures. They would be discardedas "coliforms" and might not even be tested foranaerogenesis, (which might be considered asevidence of a possible hybrid nature) except ifisolated from cases of infantile diarrhea. Indeed,several of the "coliforms" supposedly responsiblefor diarrhea are related antigenically to someShigellas (Edwards and Ewing, 1955) and may behybrid strains. Also, for example, a coliform or-ganism antigenically related to S. flexneri type 2was isolated from the stool of a patient who alsoyielded a typical, stable S. flexneri type 2 (Ewing,1953, and personal communication). A systematicsurvey for hybridizable organisms and hybridi-zation phenomena in nature, carried out withcontrolled genetic methods, would be readilyfeasible and should be fertile of remarkable find-ings and applications.A question closely related to the above dis-
cussion concerns the possible hybrid origin ofsome of the well known Shigella normotypes.Our serological findings establish that hybridiza-tion of S. flexneri type 2a or 2b with E. coli canbring about at least one major antigenic change,which is controlled by a genetic determinant nearthe Lac region of the coli-Shigella chromosome,and which produces a group of serotypes, theF22-like hybrids, similar to S. flexneri typeY. The association of the new serotype with theLac+ property is clearly a fortuitous accident ofgenetic linkage. Lac- hybrids with F22-likeserotype would not have been detected in ourcrosses. Likewise, if Lac+ hybrids of such serotypeoccurred in nature, they would probably beidentified as Shigellas only if they happened to
mutate to Lac-. In view of the finding of one suchmajor change in our limited range of experiments,it would be surprising if antigenic changes of somesort were not quite frequent following hybridiza-tion. Only a systematic study of the distributionof mating abilities among Shigella and E. colistrains in nature can help evaluate this possi-bility.The similarity of the hybrid serotype to S.
flexneri type Y raises interesting questions con-cerning the much debated significance of theY organisms. According to Boyd (1938) andothers (Edwards and Ewing, 1955) the Y strainsshould be considered as variants derived fromvarious S. ftexneri types by loss of the type anti-gens. Such variation has been observed in thelaboratory, for example, in S. flexneri types 4a(Boyd, 1938) and 2a (Ewing, 1954). Otherauthors (Weil and Saphra, 1953) consider thesevariants as different from S. flexneri type Y,which they report as possessing a type antigen ofits own.Our Y-like hybrids, derived from either 2a, 2b,
or 4a type strains, cannot be simply loss variants,because they have antigenic relations qualita-tively different from those of the parent cultures;this is especially evident for the hybrid derivedfrom S. flexneri type 2b. The genetic aspects ofthe phenomenon, namely its dependence on agenetic determinant linked with a specific regionof the genome, indicate that the serologicalchange reflects a new combination of antigen-controlling genes, probably due to the introduc-tion of coli genetic elements into Shigella. Therelation of this variation by hybridization tosimilar spontaneous variations occurring in purelines of S. flexneri remains unclarified. It ispossible that both types of genetic changes revealhidden antigenic potentialities of the strains oforigin.There seems to be no definite evidence to show
that the 0-antigens of Shigellas consists of amosaic of different antigenic determinants. Thevarious antigens are defined only in terms ofserological agglutinations and cross-adsorptiontests. If the 0-antigen (the carbohydrate-lipo-protein complex) of each organism owed itsantigenic specificity to a single molecular species(Morgan and Partridge, 1940), then the variousantigenic components, as defined by cross-re-actions, would be the expression of the differentialaffinities between the unique antigen of eachorganism, probably determined by several
genes, and the various fractions of a population ofantibody molecules that includes a whole spec-trum of configurations (Landsteiner, 1945).Each strain would react with, and could adsorbfrom, a serum those antibody molecules that fitit. The greater the proportion of such moleculesin a serum, the stronger will be the cross-reactionand the closer the inferred chemical similaritybetween the antigens of the homologous and ofthe heterologous strain. Mutation or recombi-nation of genetic determinants could give rise tonew antigens with new specificities.
If, on the other hand, the 0-antigens were amosaic of different reactive sites, then mutationor recombination could act by changing one ormore of these sites, while leaving the other un-affected. Such a mosaic structure cannot beproved by genetic tests, but only by the demon-stration of the existence of separable combiningsites for different antibody molecules in the0-antigen of a given strain, either on the intactcell or in the extracted 0-antigen complex.
SUMMARY
A number of Shigella strains were tested formating ability with Escherichia coli. All thestrains tested were fertile with F+ or Hfr de-rivatives of E. coli strain K-12 and E. coli strainC. The Shigellas behave like F- strains in thefertility system of E. coli and, like E. coli Y, canbe changed to the F+ state by mixed cultivationwith an F+ culture. Matings result in formationof hybrids, which exhibit new combinations ofcharacters typical of Shigellas with characterstypical of E. coli. The frequency of recombi-nation is lower than in similar crosses betweenstrains of E. coli, although the frequency ofmating, measured by zygotic induction ofprophage X, is comparable in both types ofcrosses. Also, some of the characters of the coliparent fail to be transmitted to the E. coli XShigella hybrids. These results suggest an in-complete genetic homology between the twogroups of organisms.Some hybrids between E. coli and type strains
of Shigella flexneri possess somatic antigensclosely related to those of S. flexneri type Y.This antigenic change is controlled by geneticfactors closely linked to factors controllinglactose utilization. The results suggest a possiblerole of hybridization occurring in nature in theevolution of the Enterobacteriaceae and in the
origin of aberrant and intermediate strains ofenteric bacteria.
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