Proc. Natl. Acad. Sci. USA Vol. 75, No. 3, pp. 1384-1388, March 1978

Cell Biology

Isolation and characterization of enterotoxin-deficient mutants of Escherichia 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, Ontario Communicated by Bernard D. Davis, December 12, 1977

The genes controlling the production of two ABSTRACT types of enterotoxin of Escherichia coli, one heat-labile (LT) and the other heat-stable (ST), are found on plasmids. The absence of a direct selection procedure has made it difficult to isolate mutants affecting toxin production. However, the availability of a naturally occurring "recombinant" plasmid, carrying genes for LT and ST formation and also for resistance to tetracycline, streptomycin, and sulfonamides, made it possible to use comutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine to enrich for such mutants. We have isolated and characterized 58 LT- mutants and 7 ST- mutants. Among the LT group we found amber mutants, temperature-sensitive mutants (most of which produce unusually heat-labile LI), and "leaky" mutants with reduced LT activity. The majority of the tested LT mutants produced immunologically crossreacting material, in most cases in wild-type amounts. Among all 17 of the LT mutants that could be transferred, the mutation was found to be on the plasmid. In contrast, only one of four transferrable ST mutants appeared to be a plasmid mutant.

mid genes for which there is no direct selection (6). The procedure has been referred to as comutagenesis (7) and is based on the known property of Ngd to induce a number of closely linked mutations within a short segment of bacterial DNA, in the vicinity of the replicating fork. Thus, if one can select for Ngd-induced mutations in a gene with a known location, one finds enrichment for mutations in neighboring genes, within a radius of about 100 kilobases (7). With the ColVBtrp plasmid, the frequency of plasmid mutants in a trp+ selected population was estimated to be about 200 times greater than in the Ngdtreated population as a whole (6). For the isolation of LT- and ST- mutants, an opportunity for comutagenesis was provided by a plasmid, pCG86, which in addition to genes for LT and ST production carries genes for resistance to three drugs, tetracycline (Tc), streptomycin (Sm), and sulfonamides (Su) (8). In order to have the proper conditions for comutagenesis, it is necessary to first isolate strains with a mutation to drug sensitivity in one of the drug resistance genes. Revertants to drug resistance can then be selected on media containing the drug. These revertants can be scored for defects in toxin production. In the present paper we describe the isolation of Tc-sensitive mutants that give a high frequency of revertants following exposure to Ngd. Among the revertants we found mutants with defective toxin production at a frequency of 1-2%. Several types of mutants, including amber nonsense mutants, temperature-sensitive mutants, "leaky" mutants with low toxin activity, and mutants with no measurable toxin activity, were obtained.

Two types of enterotoxin, one heat-labile (LT) and the other heat-stable (ST), have been found in Escherichia coli strains implicated in diarrheal disease in humans and in animals (1). The genes controlling the production of these toxins are located on plasmids (2). LT resembles cholera toxin in its mode of action (stimulation of adenylate cyclase activity) and it crossreacts immunologically 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 antigenic (4). Recently it has been shown to stimulate guanylate cylase activity in host cells (L. Graff, personal communica-

tion).

MATERIALS AND METHODS The bacterial strains and plasmids with their relevant genetic characteristics are listed in Table 1. The media used have been described 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 heart infusion broth (Difco) is a standard medium. The procedures for growing cells, testing for phenotypes, and carrying out matings have been described (11). To test for sensitivity to drugs, colonies were plated in small patches on neopeptone agar plates and replica plated onto either neopeptone or minimal agar plates containing Tc at 20 pg/ml, Sm at 20 ,g/ml, or sulfadiazine at 100,gg/ml. Isolation of Tcs Mutants. Since Tc is bacteriostatic and penicillin requires growth of the bacteria for its bactericidal

So far no plasmid mutants affecting the production of either LT or ST have been reported. This failure is largely due to the absence of direct selection procedures. The experience with diphtheria toxin (5) suggests that studies with mutants may throw light on the location and number of genes involved in toxin production, the control of the transcription and translation of these genes, and the further processing of the toxin molecules in their passage through the inner and outer cell membranes. Chain-terminating mutants, such as nonsense mutants, may give information about the organization of the toxin molecules, such as identifying the part involved in binding to the cell surface and the part responsible for stimulating adenylate cyclase activity. In the present paper we describe the isolation of mutants affecting the production of LT and of ST. We used an enrichment procedure for mutants involving mutagenesis with Nmethy-N'-nitro-N-nitrosoguanidine (Ngd). This method has been shown to be effective for the isolation of mutants in plas-

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, immunologically 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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "'advertsement in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1384

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

Cell Biology: Silva et al. Designation

Strains KL320 MA335

Table 1. Description of E. coli K12 strains and plasmids Relevant characteristics* pro his trp met strA rpsE' pro his trp met strA+ rpsE

LS289 MA373

pro his trp ilv strA+ pro his trp ilv strA supD

MA374

pro his trp ilv strA+ supF

MA3170 MA3299 MA3297 MA3298 MA3301

KL320 (pCG86) LS289 (pCG86) MA335 (pCG86) MA335 (pMS18) LS289 (pMS18)

-

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Origin

B. Bachmann (CGSC 4352) P1 transduction from a SmSSpR strain with selection for spectinomycin resistance B. Bachmann (CGSC 4385) P1 transduction of LS289 from supD strain with selection for Trp+His+ PI transduction of LS289 from supF strain with selection for Trp+His+ Mating of KL320 with strain 86 (8) Mating of MA3170 with LS289 Mating of MA3299 with MA335 Mating of MA3301 with MA335 From KL320 (pMS18)

LT+ST+ plasmids (8) TcRSMRSuRTra+ pCG86 Ngd mutagenesis of MA3170 TcSSmRSuRTrapMS12 Ngd mutagenesis of MA3170 TcSSmRSuRTrapMS15 Ngd mutagenesis of MA3170 TcSSmRSuRTra+ pMS16 Ngd mutagenesis of MA3170 TcSSmRSuRTra+ pMS18 Ngd mutagenesis of MA3170 TcSSmRSuRTra? pMS21 Ngd mutagenesis of KL320 (pMS16) TcRSmRSuSTra+ pMS28 * 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 and penicillin. For the mutagenesis we followed the procedure of Koyama et al. (6). A single colony was suspended in 5 ml of minimal medium and incubated with shaking for 6 hr at 320, prior to exposure to 15 ug of Ngd per ml for 15 min. After centrifugation and washing with minimal medium, the bacteria were suspended in 2 ml of minimal medium and 0.5-ml aliquots were added to 3 ml of either minimal medium or neopeptone broth. These cultures were incubated with shaking for 2 hr at 370; then penicillin and Tc were added, the former to a concentration of 1000 M(g/ml, the latter to 20 gg/ml. Incubation was continued for 16 hr before 0. 1-ml aliquots were plated on neopeptone agar. The plates were incubated at 370 for 1-2 days, and colonies appearing on these plates were tested for sensitivity to Tc. Isolation of LT and ST Mutants. For mutagenesis with Ngd we used the procedure described by Adelberg et al. (14). From an overnight neopeptone culture 0.1 ml was inoculated into 10 ml of fresh neopeptone broth and incubated with shaking for 3 hr at 370. The bacteria were centrifuged and resuspended in 4.5 ml of Tris-maleic buffer, pH 6.0, containing 200 gg of Ngd per ml. After 30 min of incubation at 370 they were centrifuged, washed with g-Syncase medium, and resuspended in 5 ml of this medium. To 5 ml of fresh g-Syncase medium, 0.5 ml of the mutagenized suspension was added and the culture was incubated with shaking for 16 hr at 37'. Then 0.1-ml aliquots were plated on neopeptone agar plates containing 20MAg of Tc per ml or, for selection of Su11 mutants, they were plated on minimal agar plates containing 100 ,ug of sulfadiazine per ml. After incubation of 370, colonies appearing on these plates were purified and tested for toxin production and other plasmid-controlled traits. Assay of Enterotoxins. For the assay of LT we used the Y1 mouse adrenal tumor cell system of Donta et al. (15) as modified for microtiter plates by Sack and Sack (16). Toxin production was recorded in terms of the percent rounding of cells after overnight incubation of the assay plates at 370. A rough quantitative 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 production in whole cells, 0.5-ml cultures of the bacteria were grown in g-Syncase medium for 24 hr (incubation at 370 or 420) or for 48 hr (incubation at 300). The amount of bacterial culture added to Y1 cells in each well was 0.05 ml in a total volume of 0.1 ml. To determine LT in sonic extracts, 50-ml cultures were grown in g-Syncase medium for 24 hr at 300, 370, or 420. The bacteria were harvested by centrifugation, resuspended in 3 ml of 33 mM Tris buffer, pH 7.1, and treated with a Branson S-125 Sonifier for 30 sec in the cold. The disrupted cells were centrifuged for 5 min at 40 in an Eppendorf Model 5412 centrifuge. Again, 0.05 ml of the supematant was assayed in a total volume of 0.1 ml. Since sonic extracts were usually more active than whole cultures, three 5-fold serial dilutions prepared in tissue culture medium had to be assayed in order to be within the 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 brain heart infusion broth for 16-18 hr at 370. For the assay of ST the cultures were heated at 800 for 30 min prior to testing. Strains that were scored as ST- were tested twice more and, if consistently negative, were tested again after growth in soft agar (18) and also in preparations concentrated with acetone (19). For the last-mentioned preparations, the infant mouse assay (20) was used in addition to the intestinal loop assay. Only strains that were negative in all these tests are listed as ST-. RESULTS For purposes of orientation the scheme for the isolation of plasmid mutants is shown in Fig. 1. The map of plasmid pCG86 in this figure is based on electron microscopic studies of heteroduplex molecules formed between pCG86 and derivatives of pCG86 carrying deletions for TcR and for LT+ and STI+, as well as heteroduplex molecules formed between pCG86 and plasmids of the FI incompatibility group (unpublished observations).

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Nail. Acad. Sci. USA 75 (1978)

Table 2. Isolation of LT- and ST- mutants

Exp. 1 2 3

No. of resistant mutants Selection tested

Tcs-TcR SuS-SuR

Tcs-TcR

1197 170 2000

LT-

ST-

13 2 43

ND ND 7*

SmS ND ND 25

SuS

TcS

39 ND 25

ND 3 ND

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

Ngd

Ngd TcR TcS FIG. 1. Diagram of plasmid pCG86 and scheme for localized mutagenesis with Ngd. The segments bounded by solid triangles have been deleted in plasmids generated by transduction with phage P1. Strains with plasmids deleted for segment a are Tcs, strains deleted for segment b are LT- ST-. The thicker lines within these segments represent inverted repeats. The lengths of the inverted repeats in

segment a and the DNA sequence between them are similar to those reported for the Tc transposon (21). The Tra+ (conjugal transfer ability) segment represents a region of homology with the F plasmid with coordinates 63F to 91F (22). The location of the genes for SmR and SuR is in the region of the plasmid indicated, but has not been determined more precisely. kb, kilobases.

Isolation of Tc-sensitive mutants Mutagenesis and enrichment for Tc-sensitive mutants by growth in minimal medium or in neopeptone broth in the presence of Tc and penicillin were carried out as described in Materials and Methods. The strains harboring plasmid pCG86 were multiauxotrophic derivatives of E. coli K12 free of known amber suppressors (Table 1). This permits subsequent detection of nonsense mutants in plasmid genes by transferring the mutant plasmid into strains with nonsense suppressors. From the culture grown in minimal -medium 3 of 330 colonies tested were Tcs; from the neopeptone broth culture, 5 of 28 were TcS. Five of these eight Tcs mutants gave a large increase of TcR revertants upon exposure to Ngd. These five were used in the following mutagenesis experiments. Isolation of LT- and ST- mutants Prior to the present studies we had unsuccessfully tried to isolate LT- mutants from strain 711(P307), a derivative of E. coli K12 that carries a plasmid with genes for LT and ST production but not for drug resistance (23). When plasmid pCG86 and the opportunity for comutagenesis became available, we first tested the efficiency of this procedure by comparing the frequency of SuS and SmS mutants among TcR-selected revertants with that in the Ngd-treated population as a whole. As parent strain, we used MA3298 (Table 1). Among 500 colonies selected for Tc resistance we found 17 Sms and 4 Sus mutants, whereas among 1000 unselected colonies we found no Sms or SuS mutants. On the other hand, there were auxotrophic mutants in both groups, 12 among the 500 TcR revertants and 17 among the unselected colonies. It was thus clear that selection for a

plasmid mutation led to enrichment for other plasmid mutations. We then looked for LT- and ST- mutants. Results of three experiments carried out so far are shown in Table 2. Exp. I was our first experiment; here all five Tcs plasmids were used. There was no difference among the five in the frequency of LTmutants or mutants in other plasmid genes. In Exp. 2 a Sus mutant isolated in Exp. 1 was mutagenized; the frequency of plasmid mutants among Ngd-induced SuR revertants was found to be similar to that in Exp. 1. In these two experiments the host strain for the plasmids, KL320, was SmR due to a chromosomal mutation, and we could therefore not test for Sms plasmid mutants. In Exp. 3, the host strain, MA335, was a SmS derivative of strain KL320. This is our most recent and extensive experiment and it contains some features in addition to those of the previous two experiments, which will be described. Two 10-ml cultures of strain MA3298 were grown and mutagenized with Ngd. Each of the mutagenized suspensions was distributed into 10 tubes prior to overnight growth in g-Syncase medium in order to minimize the isolation of duplicate mutants. Aliquots of 0.1 ml from the 20 tubes were plated on neopeptone agar plates containing 20 ,gg of Tc per ml. After 2 days incubation, there were about 300 colonies per plate. From each plate 100 colonies were picked and purified by single colony isolation on the same medium. The total of 2000 strains were then tested for resistance to the other drugs by replica plating and for LT production by the Y1 cell culture assay after growth of the bacteria at 420. The elevated temperature was chosen to permit subsequent detection of temperature-sensitive mutants. Later 1000 strains were also tested for ST production by the intestinal loop assay. In this experiment 43 LT- and 7 ST- mutants were obtained. The frequency of ST- mutants was lower than that of LTmutants, but the assays for ST were done 3 months after the other tests and at that time some of the TcR revertants were no longer viable. All strains that were Tox- (either LT- or ST-) on first scoring were tested repeatedly and poor growers were discarded. Thus, during the first scoring of Exp. 3 there were 150 LT- mutants. Some of the finally established mutants were double mutants for the scored traits: four LT-Sus, two SmSSuS1 one ST-Sus, and one ST-LT-. Among the 2000 TcR revertants, 435 had additional auxotrophic requirements. This is in the range of previously reported frequencies for Ngd-induced auxotrophic mutations. From the distribution of the 43 LTmutants among the 20 cultures and from differences among mutants from the same culture, it can be deduced that at least 29 of the 43 mutants are of independent origin. Although the LT- mutants isolated in Exp. 3 have not yet been tested by the ileal loop method, such tests have been done on the LT- mutants from Exp. 1 and 2. Of the 15 mutants, 14 were negative in the test. The one positive strain was also positive when sonic extracts (see below) were tested in the Y1 cell assay. Absence of activity in the Y1 cell assay can therefore be

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

Cell Biology: Silva et al. Table 3. Heat lability of LT in extracts of temperature-sensitive LT- mutants* LS289 host (new) MA335 host (original) Hr at 420 pMS123 pMS125 pCG86 pMS123 pMS125 pCG86 0 1 3 5

+++ ++

+++ ++

+++ +++

+ +

i i

++ ++

+++ +

+++ +

+++ +++

i++ ++ -

* Residual LT activity is given. pMS123 and pMS125 are temperature-sensitive LT- mutant plasmids obtained from Exp. 3, Table 2.

considered to be a reliable measure of a defect in LT production. Characterization of LT- and ST mutants The 58 LT- mutants obtained so far have been characterized further in regard to the location (on the plasmid or on the chromosome) and nature of the mutation. Ngd gives rise mainly to single base pair substitution mutants, either of the missense or the nonsense type (24). Besides LT- nonsense mutants one might therefore expect to find missense mutants which, as a result of production of an altered protein with a single amino acid 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 17 of the 58 LT- mutants were still transfer proficient. With all of these the LT phenotype was retained after transfer to strain LS289; thus the LT- mutation had occurred on the plasmid. The high incidence of Tra- (conjugal transfer ability) mutants among the LT- strains presumably reflects the large opportunity for Tra- mutations due to the presence on the plasmid of at 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 mutants gave rise to offspring with LT activity after transfer of the plasmid into the Su+ hosts and are thus amber mutants. Of the 58 LT- mutants 13 were temperature sensitive, producing LT during growth at 300 but not at 420. Of these, 12 produced an LT that is more heat labile than that produced by the wild type. To test for heat lability, we exposed sonic extracts of strains grown at 300 to 420. The remaining strain produced an LT at 300 with the same lability as the strain carrying the parental pCG86 plasmid, but did not produce LT at 420. Of the 12 strains with labile LT, 4 were Tra+ and thus temperature sensitivity could be tested after transfer to another host strain. The resulting progeny showed the same temperature sensitivity as the parental strains. Moreover, LTs formed at 300 had the same 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 LT produced at 300 by these mutants is about the same as that produced by the parental strain. This was verified by testing three serial dilutions of the LT preparations. From the results shown in Table 3 it can be inferred that these mutations to temperature sensitivity have occurred in the structural gene for LT. Thus these findings affirm the notion that the structural gene 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 provide a simple means for obtaining concentrated LT preparations. With strains carrying the wild-type plasmid we find about 100 times more LT activity in sonic extracts than we find in whole

1387

cultures or in culture filtrates. Of the 45 non-temperaturesensitive LT mutants, 11 had some LT activity in sonic extracts but not in whole cultures. With most of these strains this activity was considerably less than that found in strains with the parental pCG86 plasmid. Further evidence that these leaky mutants produce 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- character after transfer and therefore the other three presumably carry chromosomal ST- mutations. This is in contrast to the 17 Tra+ LT- mutants which all retained the LT- phenotype after transfer to another host. DISCUSSION In this paper we used a method for the enrichment of enterotoxin-deficient mutants that makes it relatively easy to isolate large numbers of such mutants. We did not determine the actual increase in the frequency of plasmid mutants due to the comutagenesis procedure, but Koyama et al. (6) estimated the increase for Tra- mutants to be about 200-fold. In our experiments the frequency of each type of plasmid mutant among colonies selected for drug resistance was about 2%. In the experiments of Adelberg et al. (14), which were done under similar conditions, the frequency of valine-resistant mutants per locus in the absence of comutagenesis can be estimated to be about 0.02%. Comparison of these figures leads to an estimate of a 100-fold enrichment per gene. The fact that comutagenesis appears to be an effective means of enriching for plasmid mutants, as shown by our failure to isolate plasmid mutants in the absence of comutagenesis implies that Ngd as used here induces clusters of plasmid mutations in only a small fraction of the treated population. On the basis of a previous study of His+ reversions (24), we expect to find 80% missense mutants and 20% nonsense mutants. Of 17 LT- mutants that could be tested, 2 were of the nonsense type. Presumably the 12 temperature-sensitive LTmutants that produce an excessively heat-labile LT are of the missense type. The nature of our LT- mutants is now being investigated further by testing them for the production of immunologically cross-reacting material (CRM). This is being done in collaboration with M. G. Bramucci and R. K. Holmes, who have developed a sensitive quantitative radioimmunoassay for LT. In preliminary experiments 17 of 24 non-temperature-sensitive LT- mutants tested and all of 6 temperaturesensitive mutants tested produced CRM. In most of these CRM+ strains, the amount of immunologically active material produced in culture filtrates was about the same as that produced by the LT+ wild type. Details of these experiments will be published subsequently. Of special interest is the finding that 9 of the 11 leaky mutants produce wild-type amounts of immunologically active material in culture filtrates. The finding of leaky LT- mutants with measurable LT activity in concentrated extracts, but not in culture filtrates, could have been interpreted in other ways besides production of an altered protein with low specific activity: e.g., a reduced rate of synthesis of normal LT or failure of the LT molecules to pass through the membranes to the outside. However, the finding of normal amounts of CRM in culture filtrates rules out these two alternate possibilities. It is of interest that among the small number of ST mutants isolated we find both a plasmid and a chromosomal location of the mutations. In contrast, all our LT- mutants that could be tested were plasmid mutants. Our results show that two or more genes control the production of ST. This difference between

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ST- and LT- may reflect the difference in their chemical constitution. ST is a relatively small molecule and, in contrast to LT, is presumably not the direct product of a structural gene. It is not known how ST is formed: either by degradation of a precursor macromolecule or by a process of assembly from smaller building blocks that does not use a nucleic acid template. Either mechanism presumably entails several steps, and this is consistent with our finding of more than one gene controlling ST production. The excellent technical assistance of Mrs. H. McKeon is gratefully acknowledged. This investigation was supported by U.S. Public Health Service Grant AI-09079 from the National Institute of Allergy and Infectious Diseases and by National Science Foundation Grant OIP74-03192-AOl. M.L.M.S. was supported by a fellowship from Coordena9io de Aperfeigoamento de Pessoal de Nivel Superior, Brasilia, D.F., Brazil, and is a faculty member of the Department of Microbiology, Escola Paulista de Medicina, Sao Paulo, Brazil. W.K.M. is the holder of U.S. Public Health Service Career Award K6 GM-15, 129, from the National Institute of General Medical Sciences. 1. Sack, R. B. (1975) Annu. Rev. Microbiol. 29, 33-353. 2. Gyles, C., So, M. & Falkow, S. (1974) J. Infect. Dis. 130, 4049. 3. Gyles, C. L. (1974) Infect. Immun. 9,564-570. 4. Jacks, T. M. & Wu, B. J. (1974) Infect. Immun. 9,342-347. 5. Pappenheimer, A. M. (1977) ANNU/ Rev. Biochem. 46, 6994. 6. Koyama, A. H., Wada, C., Nagata, T. & Yura, T. (1975) J. Bacteriol. 122, 73-79. 7. Oeschger, M. P. & Berlyn, M. K. B. (1974) Mol. Gen. Genet. 134, 77-83.

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8. Gyles, C. L., Palchaudhuri, S. & Maas, W. K. (1977) Science 198, 198-199. 9. Demerec, M., Adelberg, E. A., Clark, A. J. & Hartman, P. E. (1966) Genetics 54,61-76. 10. Bachmann, B. J., Low, K. B. & Taylor, A. L. (1976) Bacteriol. Rev.

40,116-167. 11. Dubnau, E. & Maas, W. K. (1968) J. Bacteriol. 95,531-539. 12. Evans, D. J., Evans, D. C. & Corbach, S. L (1973) Infect. Immun.

8,725-730. 13. Sack, R. B., Gorbach, S. L., Banwell, J. G., Jacobs, B., Chatterjee, D. & Mitra, R. C. (1971) J. Infect. Dis. 123,378-385. 14. Adelberg, E. A., Mandel, M. & Chen, G. C. C. (1965) Biochem. Biophys. Res. Commun. 18,788-795. 15. Donta, S. T., Moon, H. W. & Whipp, S. C. (1974) Science 183, 334-36. 16. Sack, D. A. & Sack, B. R. (19'75) Infect. Immun. 11, 334-336. 17. Gyles, C. L. & Barnum, D. A. (1969) J. Infect. Dis. 120, 419426. 18. Smith, H. W. & Halls, S. (1967) J. Pathol. Bacteriol. 93,531543. 19. Klipstein, F. A., Lee, C. S. & Engert, R. F. (1976) Infect. Immun. 14, 1004-1010. 20. Dean, A. G., Ching, Y. C., Williams, R. G. & Harden, L. B. (1972) J. Infect. Dis. 125, 407-411. 21. Kleckner, N. (1977) Cell 11, 11-23. 22. Sharp, P. A., Hsu, M. T., Ohtsubo, E. & Davidson, N (1972) J. Mol. Biol. 71,471-497. 23. Santos, D. S., Palchaudhuri, S. & Maas, W. K. (1975) J. Bacteriol. 124, 1240-1247. 24. Hartman, P. E., Hartman, Z. & Stahl, R. C. (1971) in Advances in Genetics, ed. Caspari, E W. (Academic Press, New York), Vol. 16, pp. 1-34.

Isolation and characterization of enterotoxin-deficient mutants of Escherichia coli.

Proc. Natl. Acad. Sci. USA Vol. 75, No. 3, pp. 1384-1388, March 1978 Cell Biology Isolation and characterization of enterotoxin-deficient mutants of...
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