Proc. Natl. Acad. Sci. USAVol. 75, No. 3, pp. 1384-1388, March 1978Cell Biology

Isolation and characterization of enterotoxin-deficient mutants ofEscherichia coli

(plasmid genetics/mutant enrichment/drug-resistant pathogens/altered mutant molecules/toxin immunology)

M. L. M. SILVA*t, W. K. MAAS*tt, AND C. L. GYLES§* Department of Microbiology, New York University School of Medicine, New York, New York 10016; and § Ontario Veterinary College, University of Guelph,Guelph, OntarioCommunicated by Bernard D. Davis, December 12, 1977

ABSTRACT The genes controlling the production of twotypes of enterotoxin of Escherichia coli, one heat-labile (LT) andthe other heat-stable (ST), are found on plasmids. The absenceof a direct selection procedure has made it difficult to isolatemutants affecting toxin production. However, the availabilityof a naturally occurring "recombinant" plasmid, carrying genesfor LT and ST formation and also for resistance to tetracycline,streptomycin, and sulfonamides, made it possible to use co-mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine toenrich for such mutants. We have isolated and characterized58 LT- mutants and 7 ST- mutants. Among the LT group wefound amber mutants, temperature-sensitive mutants (most ofwhich produce unusually heat-labile LI), and "leaky" mutantswith reduced LT activity. The majority of the tested LT mu-tants produced immunologically crossreacting material, in mostcases in wild-type amounts. Among all 17 of the LT mutantsthat could be transferred, the mutation was found to be on theplasmid. In contrast, only one of four transferrable ST mutantsappeared to be a plasmid mutant.

Two types of enterotoxin, one heat-labile (LT) and the otherheat-stable (ST), have been found in Escherichia coli strainsimplicated in diarrheal disease in humans and in animals (1).The genes controlling the production of these toxins are locatedon plasmids (2). LT resembles cholera toxin in its mode of action(stimulation of adenylate cyclase activity) and it crossreactsimmunologically with cholera toxin (3). Unlike cholera toxin,its chemistry has not been elucidated. ST is a smaller molecule,with a molecular weight of less than 10,000, and it is not anti-genic (4). Recently it has been shown to stimulate guanylatecylase activity in host cells (L. Graff, personal communica-tion).

So far no plasmid mutants affecting the production of eitherLT or ST have been reported. This failure is largely due to theabsence of direct selection procedures. The experience withdiphtheria toxin (5) suggests that studies with mutants maythrow light on the location and number of genes involved intoxin production, the control of the transcription and translationof these genes, and the further processing of the toxin moleculesin their passage through the inner and outer cell membranes.Chain-terminating mutants, such as nonsense mutants, maygive information about the organization of the toxin molecules,such as identifying the part involved in binding to the cellsurface and the part responsible for stimulating adenylate cy-clase activity.

In the present paper we describe the isolation of mutantsaffecting the production of LT and of ST. We used an enrich-ment procedure for mutants involving mutagenesis with N-methy-N'-nitro-N-nitrosoguanidine (Ngd). This method hasbeen shown to be effective for the isolation of mutants in plas-

mid genes for which there is no direct selection (6). The pro-cedure has been referred to as comutagenesis (7) and is basedon the known property of Ngd to induce a number of closelylinked mutations within a short segment of bacterial DNA, inthe vicinity of the replicating fork. Thus, if one can select forNgd-induced mutations in a gene with a known location, onefinds enrichment for mutations in neighboring genes, withina radius of about 100 kilobases (7). With the ColVBtrp plasmid,the frequency of plasmid mutants in a trp+ selected populationwas estimated to be about 200 times greater than in the Ngd-treated population as a whole (6).

For the isolation of LT- and ST- mutants, an opportunityfor comutagenesis was provided by a plasmid, pCG86, whichin addition to genes for LT and ST production carries genes forresistance to three drugs, tetracycline (Tc), streptomycin (Sm),and sulfonamides (Su) (8). In order to have the proper conditionsfor comutagenesis, it is necessary to first isolate strains with amutation to drug sensitivity in one of the drug resistance genes.Revertants to drug resistance can then be selected on mediacontaining the drug. These revertants can be scored for defectsin toxin production. In the present paper we describe the iso-lation of Tc-sensitive mutants that give a high frequency ofrevertants following exposure to Ngd. Among the revertantswe found mutants with defective toxin production at a fre-quency of 1-2%. Several types of mutants, including ambernonsense mutants, temperature-sensitive mutants, "leaky"mutants with low toxin activity, and mutants with no measur-able toxin activity, were obtained.

MATERIALS AND METHODSThe bacterial strains and plasmids with their relevant geneticcharacteristics are listed in Table 1. The media used have beendescribed as follows: minimal medium and neopeptone broth(11), Evans medium (12), and Syncase medium (13) with 0.5%glucose substituted for 0.5% sucrose (g-Syncase). Brain heartinfusion broth (Difco) is a standard medium. The proceduresfor growing cells, testing for phenotypes, and carrying outmatings have been described (11). To test for sensitivity todrugs, colonies were plated in small patches on neopeptone agarplates and replica plated onto either neopeptone or minimalagar plates containing Tc at 20 pg/ml, Sm at 20 ,g/ml, or sul-fadiazine at 100,gg/ml.

Isolation of Tcs Mutants. Since Tc is bacteriostatic andpenicillin requires growth of the bacteria for its bactericidal

Abbreviations: Tc, tetracycline; Sm, streptomycin; Su, sulfonamides;Ngd, N-methyl-N'-nitro-N-nitrosoguanidine; LT, heat-labile toxin;ST, heat-stable toxin; Tra, conjugal transfer ability; CRM, immu-nologically crossreacting material.t Present address: Departamento de Microbiologia e Parasitologia,Escola Paulista de Medicina, Sao Paulo, S.P., Brazil.

* To whom reprint requests should be addressed.

1384

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Proc. Natl. Acad. Sci. USA 75 (1978) 1385

Table 1. Description of E. coli K12 strains and plasmids

Designation Relevant characteristics* Origin

StrainsKL320 pro his trp met strA rpsE' B. Bachmann (CGSC 4352)MA335 pro his trp met strA+ rpsE P1 transduction from a SmSSpR strain with selection for

spectinomycin resistanceLS289 pro his trp ilv strA+ B. Bachmann (CGSC 4385)MA373 pro his trp ilv strA supD P1 transduction of LS289 from supD strain with

selection for Trp+His+MA374 pro his trp ilv strA+ supF PI transduction of LS289 from supF strain with

selection for Trp+His+MA3170 KL320 (pCG86) - Mating of KL320 with strain 86 (8)MA3299 LS289 (pCG86) Mating of MA3170 with LS289MA3297 MA335 (pCG86) Mating of MA3299 with MA335MA3298 MA335 (pMS18) Mating of MA3301 with MA335MA3301 LS289 (pMS18) From KL320 (pMS18)

LT+ST+ plasmidspCG86 TcRSMRSuRTra+ (8)pMS12 TcSSmRSuRTra- Ngd mutagenesis of MA3170pMS15 TcSSmRSuRTra- Ngd mutagenesis of MA3170pMS16 TcSSmRSuRTra+ Ngd mutagenesis of MA3170pMS18 TcSSmRSuRTra+ Ngd mutagenesis of MA3170pMS21 TcSSmRSuRTra? Ngd mutagenesis of MA3170pMS28 TcRSmRSuSTra+ Ngd mutagenesis of KL320 (pMS16)

* Genotype symbols follow Demerec et al. (9). Explanation for symbols may be found in the review of Bachmann et al. (10) and in the text.

action, Tcs mutants should survive in the presence of Tc andpenicillin. For the mutagenesis we followed the procedure ofKoyama et al. (6). A single colony was suspended in 5 ml ofminimal medium and incubated with shaking for 6 hr at 320,prior to exposure to 15 ug of Ngd per ml for 15 min. Aftercentrifugation and washing with minimal medium, the bacteriawere suspended in 2 ml of minimal medium and 0.5-ml aliquotswere added to 3 ml of either minimal medium or neopeptonebroth. These cultures were incubated with shaking for 2 hr at370; then penicillin and Tc were added, the former to a con-centration of 1000 M(g/ml, the latter to 20 gg/ml. Incubationwas continued for 16 hr before 0. 1-ml aliquots were plated onneopeptone agar. The plates were incubated at 370 for 1-2 days,and colonies appearing on these plates were tested for sensitivityto Tc.

Isolation of LT and ST Mutants. For mutagenesis withNgd we used the procedure described by Adelberg et al. (14).From an overnight neopeptone culture 0.1 ml was inoculatedinto 10 ml of fresh neopeptone broth and incubated withshaking for 3 hr at 370. The bacteria were centrifuged and re-suspended in 4.5 ml of Tris-maleic buffer, pH 6.0, containing200 gg of Ngd per ml. After 30 min of incubation at 370 theywere centrifuged, washed with g-Syncase medium, and re-suspended in 5 ml of this medium. To 5 ml of fresh g-Syncasemedium, 0.5 ml of the mutagenized suspension was added andthe culture was incubated with shaking for 16 hr at 37'. Then0.1-ml aliquots were plated on neopeptone agar plates con-taining 20MAg of Tc per ml or, for selection of Su11 mutants, theywere plated on minimal agar plates containing 100 ,ug of sul-fadiazine per ml. After incubation of 370, colonies appearingon these plates were purified and tested for toxin productionand other plasmid-controlled traits.

Assay of Enterotoxins. For the assay of LT we used the Y1mouse adrenal tumor cell system of Donta et al. (15) as modi-fied for microtiter plates by Sack and Sack (16). Toxin pro-duction was recorded in terms of the percent rounding of cellsafter overnight incubation of the assay plates at 370. A roughquantitative estimation of LT was made as follows: +++ =

80-100% of cells rounded, ++ = 50-80% rounded, + = 20-W0%rounded, and ± = 10-20% rounded. To determine LT pro-duction in whole cells, 0.5-ml cultures of the bacteria weregrown in g-Syncase medium for 24 hr (incubation at 370 or 420)or for 48 hr (incubation at 300). The amount of bacterial cultureadded to Y1 cells in each well was 0.05 ml in a total volume of0.1 ml. To determine LT in sonic extracts, 50-ml cultures weregrown in g-Syncase medium for 24 hr at 300, 370, or 420. Thebacteria were harvested by centrifugation, resuspended in 3ml of 33 mM Tris buffer, pH 7.1, and treated with a BransonS-125 Sonifier for 30 sec in the cold. The disrupted cells werecentrifuged for 5 min at 40 in an Eppendorf Model 5412 cen-trifuge. Again, 0.05 ml of the supematant was assayed in a totalvolume of 0.1 ml. Since sonic extracts were usually more activethan whole cultures, three 5-fold serial dilutions prepared intissue culture medium had to be assayed in order to be withinthe sensitive range of the assay.-LT and ST were also assayed for fluid accumulation in ligated

segments of pig intestine (17). The bacteria were grown in brainheart infusion broth for 16-18 hr at 370. For the assay of ST thecultures were heated at 800 for 30 min prior to testing. Strainsthat were scored as ST- were tested twice more and, if consis-tently negative, were tested again after growth in soft agar (18)and also in preparations concentrated with acetone (19). Forthe last-mentioned preparations, the infant mouse assay (20)was used in addition to the intestinal loop assay. Only strainsthat were negative in all these tests are listed as ST-.

RESULTS

For purposes of orientation the scheme for the isolation ofplasmid mutants is shown in Fig. 1. The map of plasmid pCG86in this figure is based on electron microscopic studies ofheteroduplex molecules formed between pCG86 and deriva-tives of pCG86 carrying deletions for TcR and for LT+ and STI+,as well as heteroduplex molecules formed between pCG86 andplasmids of the FI incompatibility group (unpublished obser-vations).

Cell Biology: Silva et al.

Proc. Nail. Acad. Sci. USA 75 (1978)

Ngd

TcS Ngd TcR

FIG. 1. Diagram of plasmid pCG86 and scheme for localizedmutagenesis with Ngd. The segments bounded by solid triangles havebeen deleted in plasmids generated by transduction with phage P1.Strains with plasmids deleted for segment a are Tcs, strains deletedfor segment b are LT- ST-. The thicker lines within these segmentsrepresent inverted repeats. The lengths of the inverted repeats insegment a and the DNA sequence between them are similar to thosereported for the Tc transposon (21). The Tra+ (conjugal transferability) segment represents a region of homology with the F plasmidwith coordinates 63F to 91F (22). The location of the genes for SmRand SuR is in the region of the plasmid indicated, but has not beendetermined more precisely. kb, kilobases.

Isolation of Tc-sensitive mutants

Mutagenesis and enrichment for Tc-sensitive mutants bygrowth in minimal medium or in neopeptone broth in thepresence of Tc and penicillin were carried out as described inMaterials and Methods. The strains harboring plasmid pCG86were multiauxotrophic derivatives of E. coli K12 free of knownamber suppressors (Table 1). This permits subsequent detectionof nonsense mutants in plasmid genes by transferring the mu-tant plasmid into strains with nonsense suppressors. From theculture grown in minimal-medium 3 of 330 colonies tested wereTcs; from the neopeptone broth culture, 5 of 28 were TcS. Fiveof these eight Tcs mutants gave a large increase of TcR rever-

tants upon exposure to Ngd. These five were used in the fol-lowing mutagenesis experiments.

Isolation of LT- and ST- mutants

Prior to the present studies we had unsuccessfully tried to isolateLT- mutants from strain 711(P307), a derivative of E. coli K12that carries a plasmid with genes for LT and ST production butnot for drug resistance (23). When plasmid pCG86 and theopportunity for comutagenesis became available, we first testedthe efficiency of this procedure by comparing the frequencyof SuS and SmS mutants among TcR-selected revertants withthat in the Ngd-treated population as a whole. As parent strain,we used MA3298 (Table 1). Among 500 colonies selected forTc resistance we found 17 Sms and 4 Sus mutants, whereasamong 1000 unselected colonies we found no Sms or SuS mu-tants. On the other hand, there were auxotrophic mutants inboth groups, 12 among the 500 TcR revertants and 17 amongthe unselected colonies. It was thus clear that selection for a

Table 2. Isolation of LT- and ST- mutants

No. ofresistantmutants

Exp. Selection tested LT- ST- SmS SuS TcS

1 Tcs-TcR 1197 13 ND ND 39 ND2 SuS-SuR 170 2 ND ND ND 33 Tcs-TcR 2000 43 7* 25 25 ND

ND, not done.* Among 1000 TcR mutants tested.

plasmid mutation led to enrichment for other plasmid muta-tions.We then looked for LT- and ST- mutants. Results of three

experiments carried out so far are shown in Table 2. Exp. I wasour first experiment; here all five Tcs plasmids were used. Therewas no difference among the five in the frequency of LT-mutants or mutants in other plasmid genes. In Exp. 2 a Susmutant isolated in Exp. 1 was mutagenized; the frequency ofplasmid mutants among Ngd-induced SuR revertants was foundto be similar to that in Exp. 1. In these two experiments the hoststrain for the plasmids, KL320, was SmR due to a chromosomalmutation, and we could therefore not test for Sms plasmidmutants. In Exp. 3, the host strain, MA335, was a SmS derivativeof strain KL320. This is our most recent and extensive experi-ment and it contains some features in addition to those of theprevious two experiments, which will be described.Two 10-ml cultures of strain MA3298 were grown and mu-

tagenized with Ngd. Each of the mutagenized suspensions wasdistributed into 10 tubes prior to overnight growth in g-Syncasemedium in order to minimize the isolation of duplicate mutants.Aliquots of 0.1 ml from the 20 tubes were plated on neopeptoneagar plates containing 20 ,gg of Tc per ml. After 2 days incu-bation, there were about 300 colonies per plate. From each plate100 colonies were picked and purified by single colony isolationon the same medium. The total of 2000 strains were then testedfor resistance to the other drugs by replica plating and for LTproduction by the Y1 cell culture assay after growth of thebacteria at 420. The elevated temperature was chosen to permitsubsequent detection of temperature-sensitive mutants. Later1000 strains were also tested for ST production by the intestinalloop assay.

In this experiment 43 LT- and 7 ST- mutants were obtained.The frequency of ST- mutants was lower than that of LT-mutants, but the assays for ST were done 3 months after theother tests and at that time some of the TcR revertants were nolonger viable. All strains that were Tox- (either LT- or ST-)on first scoring were tested repeatedly and poor growers werediscarded. Thus, during the first scoring of Exp. 3 there were150 LT- mutants. Some of the finally established mutants weredouble mutants for the scored traits: four LT-Sus, two SmSSuS1one ST-Sus, and one ST-LT-. Among the 2000 TcR revertants,435 had additional auxotrophic requirements. This is in therange of previously reported frequencies for Ngd-inducedauxotrophic mutations. From the distribution of the 43 LT-mutants among the 20 cultures and from differences amongmutants from the same culture, it can be deduced that at least29 of the 43 mutants are of independent origin.

Although the LT- mutants isolated in Exp. 3 have not yetbeen tested by the ileal loop method, such tests have been doneon the LT- mutants from Exp. 1 and 2. Of the 15 mutants, 14were negative in the test. The one positive strain was also pos-itive when sonic extracts (see below) were tested in the Y1 cellassay. Absence of activity in the Y1 cell assay can therefore be

1386 Cell Biology: Silva et at.

Proc. Natl. Acad. Sci. USA 75 (1978) 1387

Table 3. Heat lability of LT in extracts of temperature-sensitiveLT- mutants*

Hr at MA335 host (original) LS289 host (new)420 pMS123 pMS125 pCG86 pMS123 pMS125 pCG86

0 +++ +++ +++ +++ +++ +++1 ++ ++ +++ + + +++3 + i ++ i++5 + i ++ - - ++

* Residual LT activity is given. pMS123 and pMS125 are tempera-ture-sensitive LT- mutant plasmids obtained from Exp. 3, Table2.

considered to be a reliable measure of a defect in LT produc-tion.

Characterization of LT- and ST mutantsThe 58 LT- mutants obtained so far have been characterizedfurther in regard to the location (on the plasmid or on thechromosome) and nature of the mutation. Ngd gives rise mainlyto single base pair substitution mutants, either of the missenseor the nonsense type (24). Besides LT- nonsense mutants onemight therefore expect to find missense mutants which, as aresult of production of an altered protein with a single aminoacid change, are phenotypically temperature sensitive or are"leaky," having low but measureable LT activity.To test for plasmid location of the mutation it is necessary to

transfer the plasmid to another host strain. However, only 17of the 58 LT- mutants were still transfer proficient. With allof these the LT phenotype was retained after transfer to strainLS289; thus the LT- mutation had occurred on the plasmid.The high incidence of Tra- (conjugal transfer ability) mutantsamong the LT- strains presumably reflects the large opportu-nity for Tra- mutations due to the presence on the plasmid ofat least 16 genes controlling conjugal transfer.To test for the presence of a nonsense LT- mutation, the 17

Tra+ strains were mated with strains MA373 and MA374,carrying amber suppressors D and F, respectively. Two mutantsgave rise to offspring with LT activity after transfer of theplasmid into the Su+ hosts and are thus amber mutants.Of the 58 LT- mutants 13 were temperature sensitive, pro-

ducing LT during growth at 300 but not at 420. Of these, 12produced an LT that is more heat labile than that produced bythe wild type. To test for heat lability, we exposed sonic extractsof strains grown at 300 to 420. The remaining strain producedan LT at 300 with the same lability as the strain carrying theparental pCG86 plasmid, but did not produce LT at 420. Ofthe 12 strains with labile LT, 4 were Tra+ and thus temperaturesensitivity could be tested after transfer to another host strain.The resulting progeny showed the same temperature sensitivityas the parental strains. Moreover, LTs formed at 300 had thesame heat lability at 420 in the new host as in the original one.This is shown for two mutants in Table 3. The amount of LTproduced at 300 by these mutants is about the same as thatproduced by the parental strain. This was verified by testingthree serial dilutions of the LT preparations. From the resultsshown in Table 3 it can be inferred that these mutations totemperature sensitivity have occurred in the structural genefor LT. Thus these findings affirm the notion that the structuralgene for LT is located on a plasmid.To test for leaky mutants that produce an LT with a low

specific activity we used sonic extracts, because they providea simple means for obtaining concentrated LT preparations.With strains carrying the wild-type plasmid we find about 100times more LT activity in sonic extracts than we find in whole

cultures or in culture filtrates. Of the 45 non-temperature-sensitive LT mutants, 11 had some LT activity in sonic extractsbut not in whole cultures. With most of these strains this activitywas considerably less than that found in strains with the parentalpCG86 plasmid. Further evidence that these leaky mutantsproduce an altered LT will be mentioned in the Discussion.The ST- mutants isolated in Exp. 3 have been tested for

conjugal transfer of the ST- phenotype. Of the seven mutants,four were Tra+. Only one of these retained the ST- characterafter transfer and therefore the other three presumably carrychromosomal ST- mutations. This is in contrast to the 17 Tra+LT- mutants which all retained the LT- phenotype aftertransfer to another host.

DISCUSSIONIn this paper we used a method for the enrichment of entero-toxin-deficient mutants that makes it relatively easy to isolatelarge numbers of such mutants. We did not determine the ac-tual increase in the frequency of plasmid mutants due to thecomutagenesis procedure, but Koyama et al. (6) estimated theincrease for Tra- mutants to be about 200-fold. In our experi-ments the frequency of each type of plasmid mutant amongcolonies selected for drug resistance was about 2%. In the ex-periments of Adelberg et al. (14), which were done undersimilar conditions, the frequency of valine-resistant mutantsper locus in the absence of comutagenesis can be estimated tobe about 0.02%. Comparison of these figures leads to an estimateof a 100-fold enrichment per gene. The fact that comutagenesisappears to be an effective means of enriching for plasmidmutants, as shown by our failure to isolate plasmid mutants inthe absence of comutagenesis implies that Ngd as used hereinduces clusters of plasmid mutations in only a small fractionof the treated population.On the basis of a previous study of His+ reversions (24), we

expect to find 80% missense mutants and 20% nonsense mu-tants. Of 17 LT- mutants that could be tested, 2 were of thenonsense type. Presumably the 12 temperature-sensitive LT-mutants that produce an excessively heat-labile LT are of themissense type. The nature of our LT- mutants is now beinginvestigated further by testing them for the production of im-munologically cross-reacting material (CRM). This is beingdone in collaboration with M. G. Bramucci and R. K. Holmes,who have developed a sensitive quantitative radioimmunoassayfor LT. In preliminary experiments 17 of 24 non-tempera-ture-sensitive LT- mutants tested and all of 6 temperature-sensitive mutants tested produced CRM. In most of these CRM+strains, the amount of immunologically active material pro-duced in culture filtrates was about the same as that producedby the LT+ wild type. Details of these experiments will bepublished subsequently.Of special interest is the finding that 9 of the 11 leaky mutants

produce wild-type amounts of immunologically active materialin culture filtrates. The finding of leaky LT- mutants withmeasurable LT activity in concentrated extracts, but not inculture filtrates, could have been interpreted in other waysbesides production of an altered protein with low specific ac-tivity: e.g., a reduced rate of synthesis of normal LT or failureof the LT molecules to pass through the membranes to theoutside. However, the finding of normal amounts of CRM inculture filtrates rules out these two alternate possibilities.

It is of interest that among the small number of ST mutantsisolated we find both a plasmid and a chromosomal location ofthe mutations. In contrast, all our LT- mutants that could betested were plasmid mutants. Our results show that two or moregenes control the production of ST. This difference between

Cell Biology: Silva et al.

Proc. Nati. Acad. Sci. USA 75 (1978)

ST- and LT- may reflect the difference in their chemicalconstitution. ST is a relatively small molecule and, in contrastto LT, is presumably not the direct product of a structural gene.It is not known how ST is formed: either by degradation of aprecursor macromolecule or by a process of assembly fromsmaller building blocks that does not use a nucleic acid tem-plate. Either mechanism presumably entails several steps, andthis is consistent with our finding of more than one gene con-trolling ST production.The excellent technical assistance of Mrs. H. McKeon is gratefully

acknowledged. This investigation was supported by U.S. Public HealthService Grant AI-09079 from the National Institute of Allergy andInfectious Diseases and by National Science Foundation Grant OIP-74-03192-AOl. M.L.M.S. was supported by a fellowship from Coor-dena9io de Aperfeigoamento de Pessoal de Nivel Superior, Brasilia,D.F., Brazil, and is a faculty member of the Department of Micro-biology, Escola Paulista de Medicina, Sao Paulo, Brazil. W.K.M. is theholder of U.S. Public Health Service Career Award K6 GM-15, 129,from the National Institute of General Medical Sciences.

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