repeating "O units" whose synthesis is determined carrier lipid, ACL, in Salmonella groups B, D

Transcription

1 JOURNAL OF BACTERIOLOGY, Sept. 1974, p Copyright American Society for Microbiology Vol. 119, No. 3 Printed in U.S.A. Participation of Lipopolysaccharide Genes in the Determination of the Enterobacterial Common Antigen: Analysis in Salmonella Groups B and C1 P. HELENA MAKELA AND H. MAYER Central Public Health Laboratory (State Serum Institute), Helsinki, Finland, and Max-Planck-Institut fur Immunbiologie, Freiburg, Germany Received for publication 29 March 1974 The enterobacterial common antigen (CA) is present in salmonellae of groups B (S. typhimurium) and C1 (S. montevideo). Mutation at the rfe gene(s), which is required for the biosynthesis of 0 side chains of the lipopolysaccharide in group C1 (S-6,7) but not in group B (S-4, 12), destroys the capacity of the bacteria to synthesize CA. When such mutated group C1 rfe genes (C-rfe-) were introduced into group B strains, the hybrids also became CA- and could be restored to CA+ by introduction of either C-rfe+ or B-rfe+ (corresponding genetic region in group B). This indicated the presence of genes for CA synthesis. at the rfe site in both groups B and C1. In rfe- mutants of group C1, which were rough and CA-, the CA+ phenotype could be restored by replacing the rfe- gene(s) with C-rfe+. In contrast, B-rfe+ was able to support the synthesis of trace amounts of CA only, although it was sufficient to restore their ability to synthesize the S-6,7 side chain of the lipopolysaccharide. Corresponding hybrids (B-rfe+, C-rfb+ or C-rfb-) were constructed by introducing the C-rfb genes into a group B strain; they also showed only a trace of CA reactivity. 765 Although the chemical nature of the enterobacterial common antigen (CA; 5, 7) is not known, there are reasons to suspect that sugar moieties are a part of the CA (2, 3). In this case the biosynthesis of CA might share precursors or pathways, or both, with the biosynthesis of the lipopolysaccharide (LPS), a major component of the outer layer of the cell wall. This possibility prompted us to study sets of previously well-characterized LPS mutants and determine whether they still can synthesize CA. A study of R mutants of Salmonella minnesota (O group L) implicated the rfe genes as being required for CA synthesis (9). We now have confirmed and extended this finding in a study of S. montevideo (O group Cl) and S. typhimurium (O group B) strains. In addition to the rfe gene(s), a function determined by the rfb cluster of S. typhimurium also appears to be necessary for CA synthesis. To facilitate the following discussion, it seems necessary to give a short description of the genes involved in the biosynthesis of LPS (Fig. 1 and 2; 10, 17). The core is, as far as is known, the same in all Salmonella serogroups. It is synthesized under the direction of rfa genes. The side chains of the LPS vary greatly in structure, and the 0 antigenic specificities of different Salmonella serogroups are based on their structure. These 0 side chains are polymers of repeating "O units" whose synthesis is determined by the rfb cluster of genes. The 0- specific units are synthesized separately on a carrier lipid, ACL, in Salmonella groups B, D and E. The rfb genes contain all the information needed for the synthesis of the "O-specific" sugars and for their assembly into 0 units. Thus, a set of rfb genes introduced from group B (here specified as B-rfb) into a group C1 organism directs the synthesis of 0-specific units of the sort found in group B, described by the 0-antigenic formula 4,12. Also, C-rfb genes introduced into a B group organism determine the synthesis of the 0 side chain with the formula 6,7 typical of group C1. The polymerization of the 0 units of the group B type to a smooth, S-4, 12 LPS requires the function of a separate rfc gene(s). Therefore, the group B and C1 strains mentioned above (that have received B-rfb+ but not B-rfc+) have a defective LPS, SR-4, 12, with only one 0 unit in each side chain (instead of the normal length of 4 to 30 units). An allele of rfc is not known in group C, and is not required for the synthesis of the complete LPS of type S-6, 7. The 0 polymer is attached to the core through a ligase apparently

2 766 MAKELA AND MAYER J. BACTERIOL. 0 Side chain Core Deep region LPS Chemotype Genes (O unit)n 0 unit 5 hexoses + 3 heptoses KDO, lipid A, etc. Ri = rough (R) with incomplete core Ra = R with complete core S = smooth with 0 specificity depending on the structure of the 0 unit: 4, 12 in group B (abequose - mannose - rhamnose - galactose) 6, 7 in group Cl (mannose, N-acetylglucosamine) rfb for structure of 0 units rfc in group B for polymerization of 0 units rfe in group C, for assembly of 0 side chain rfbt, rfal in group B for translocation of 0 side chain to core FIG. 1. Schematic structure of the Salmonella lipopolysaccharide (LPS) showing the rf. genes participating in its synthesis (9, 12). KDO, ketodeoxyoctanoate. met E 2). Genetic material deriving from the group C, rfe I I organism is marked, when relevant, with the prefix C;!/ and B-group material is marked with prefix B (e.g., B-rfb = rfb cluster deriving from group B). R mutants. Previously described types of R (rough; genetically rf.) mutants were used (Fig. 1). The rfb mutants have the complete LPS core of chemotype Ra. The rfe mutation, together with C-rfb+ (e.g., in group C, strains), causes a similar R phenotype with the complete Ra core (8). A rfe mutation, together with B-rfb+, allows the synthesis of a smooth (S) LPS of specificity 4,12 (manuscript in preparation). Conr fc sequently, R mutants of type Ra of strains with C-rfb fc are mutated either at the rfb cluster close to the his operon or at rfe between the loci ilv and mete, and are distinguished by genetic means. rf b' 'his In our tests, the LPS character of the strains was FIG. 2. Simplified map of the Salmoneli'la chromo- studied by slide agglutination in 4% saline and in some showing the position of LPS genes rjfa, rfb, rfc, specific rabbit antisera appropriately diluted (4), and and rfe, and the origin and direction of trarisfer of the also by sensitivity to a set of S- and R-specific phages donor Hfr strains used (15, 16). (19). Specifically, the Ra-type LPS was indicated by the sensitivity of the strain to phage Felix 01 and resistance to smooth-specific phages P22 and 9NA for determined by the joint action of a rfc zl gene in strains with the the rfa cluster and a rfbt gene in the 0 antigens 4,12, or to phage PCO1 rfb cluster. (manuscript in preparation) for 0-6,7 strains. The rfe genes have been recognized as being Genetic methods. Mutants were selected after necessary for the synthesis of the 0 sidle chain in treatment with diethylsulfate or sometimes with N- groups C1 and L; in their absence 0 units are methyl-n'-nitro-n-nitrosoguanidine, and character- (8). ized genetically by conjugation using standard found neither in LPS nor as a free "haypten" methods (7, 16). Previously described Hfr strains were MATERIALS AND METHOD! S used for conjugation (7, 16). Various R mutants were selected from these strains (8) after diethylsulfate Bacteria. Derivatives of S. montevideo Ky129 (O treatment and used to construct hybrids combining antigens 6,7) (1) were used as represeintatives of rf. loci of groups B and/or C, (7). Nearby auxotrophic group C,. Group B strains with 0 antigen' s (1), 4, (5), alleles (his for rfb; ilv and/or mete for rfe) were used 12, abbreviated as 4,12 were derivatives caf S. typhi- to select specific classes of recombinants, among murium LT2 or S. abony KylO3 (1). The genetic do- which the appropriate rf. genotypes were found (7, 8). nors were Hfr types previously described (7, 16; Fig. In some cases, co-transduction of rfb with his was rfa

3 VOL. 119, 1974 COMMON ANTIGEN IN SALMONELLA GROUPS B AND C1 used to select similar hybrids (15, 17). (A more complete description of the rf. donors and these hybrids will be published.) CA determination. The presence or absence of CA was investigated by the indirect hemagglutination test (11) as described by MbIkeli et al. (9). Erythrocytes were sensitized by using differing amounts of a saline extract of the bacteria. The antiserum used for agglutination of these sensitized cells was serum of a rabbit immunized with a Shigella boydii type 3- R mutant (18). A 0.01-ml volume of such an extract from CA+ strains could sensitize the cells to be agglutinated to maximal titer (about 5,000, corresponding to serum dilution 1:5,000). Even 1 ml of extract from CA- strains left the titer at < 10. Some strains gave a titer of <10 with 0.1 ml of extract, whereas 1 ml raised it to approximately 80; these were designated CAtra- or CAtr (9). RESULTS rfb region and CA in group B. The wildtype S. typhimurium is CA+ as are all salmonellae and other bacteria of the Enterobacteriaceae. Its derivatives, in which the rfb was manipulated in various ways, are shown in Table 1. In this table, strain type 1 is the wild-type strain with an intact, functioning rfb region of group B (B-rfb+); it is CA+. Several rfb point mutants were tested and found also to be CA+ (type 2; B-rfb-). Hybrids in which the B-rfb and his were replaced by conjugation or transduction (7, 17) with the corresponding group C1 material were analyzed next. Strains of type 3 have an intact, functional C-rfb+, as shown by the fact that their LPS is S-6, 7; they contain only trace amounts of CA (Table 1). In strains of type 4, the C-rfb contained a (presumed) point mutation, and the LPS was of type Ra; these were likewise CAtr. Finally, in type 5 strains the C-rfb region was apparently damaged through crossovers within rfb, as judged from their loss of activity of several rfb enzymes (14, 17)-again they showed only trace reactivity of CA. The introduction of C-rfb into group B thus had a profound influence on the CA content of the strains. In fact, the CA activity of the hybrids was so weak that we were not sure of its specificity. We then tested their concentrated extracts and found them able to sensitize erythrocytes to the high hemagglutination titer obtained with extracts of CA+ strains. By this means it was estimated that the trace reaction corresponds to less than 5% of the normal CA content. CA in various rf. mutants in group C1. The above findings suggest a difference between groups B and C1 with respect to their genetic determinants of CA synthesis. Several other differences between these groups have been recognized in the organization of their LPS genes (see above). The requirement of rfe genes for 0 side chain synthesis in group C 1, but not in group B, pointed to the rfe locus as a possible site of CA determinants in group C 1. In fact, the presence of CA genes at the rfe locus had at this time been demonstrated in group L (9). Although mutations at rfb or rfa have no effect on the CA content of the bacteria of group C1, rfe mutants are CA- (Table 2). The LPS itself has no effect on CA; both the CA+ rfb- and CArfe- mutants have LPS of the same chemotype Ra Ċomparison of rfe and rfb loci in groups B and C1. We constructed a series of hybrids in which the rfe and rfb regions were permutated between groups B and C (Table 3). S. typhimurium strains that have received the rfe region from group C1, either intact (C-rfe+) or mutated (C-rfe-), nevertheless make a normal LPS with 0-4,12 side chains. (The rfe genes are required for the synthesis of 0 side chains of the group C1 sort [0-6,7] but not of the group B sort [0-4, 12].) The hybrid of type 10 (Table 3), which is C-rfe-, was prepared by crossing a rfe- mutant of S. montevideo (HfrH14 strain SH3862) as a donor with the group B (LT2) strain SH380, and 767 TABLE 1. Effect of the rfb region on CA in the S. typhimurium (group B) line LT2 Strain type rfb genotypea LPSb CAc Derived from Examples of strains 1 B+ S-4,12 + SH380, mete338 ilva401 his-5406 str 2 B- point mutation Ra + Mutation of 1 SL (19) 3 C+ S-6,7 Traced S. montevideo - x 1 SH4146 C-his+ C-rfb+ 4 C- point mutation Ra Traced S. montevideo - x 1 SH5243 C-his+ C-rfb C/B Ra Traced S. montevideo -x 1 SH1086 (14) a B, From group B; C, from group C1. b Ra, Rough with complete core, no 0 hapten. c At least five separate strains tested of each sort. d Weak hemagglutination titer when a large amount of sensitizing extract is used, corresponding to less than 5% of normal CA content.

4 768 MAKELA AND MAYER J. BACTrERIOL. TABLE 2. Effect of different rf. mutations on CA in Salmonella montevideo (group C,) line Ky129 Strain rf. genotype type rfe rfa rfb LPSa CA" Derived from Examples of strains S-6,7 + SW829 (8), SH1605 HfrH Ra + Mutation of 6 SH1676 C-rfb-3795 (8) 8 + _ + Ri + Mutation of 6 SH3269 C-rfa-3652 (8) Ra _ Mutation of 6 SH1675 C-rfe-3623 (8), SH3862 HfrH14 C-rfe-3853 a Ra, Rough with complete core, no 0 hapten; Ri, rough with incomplete core, 0 hapten present. At least five separate strains tested of each sort. TABLE 3. CA in strains with various combinations of rfe and rfb from groups B and/or C,a Relevant Organism Strain genotypea LPSb CAe Derived from Examples of strains type rfe rfb S. typhimurium 10 C- B+ S-4,12 - C-rfe- (CA-) -x 1 (CA') SH4145 C-rfe-3853 derivatives 11 C+ B+ S-4,12 + C-rfe+ (CA+) -x 10 (CA-) SH5834 C-rfe+ 12 B+ B+ S-4,12 + B-rfe+ (CA+) -x 10 (CA-) SH5835 B-rfe+ S. montevideo 9 C - C+ Ra _ rfe- mutation of 6 (CA+) SH1675 C-rfe-3623 derivatives 13 C + C + S-6,7 + C-rfe+ (CA+) - x 9 (CA-) SH3463 C-rfe+ 14 B+ C+ S-6,7 Traced B-rfe+ (CA+) -x 9 (CA-) SH3461 B-rfe+ a-d See footnotes a through d of Table 1. Donor strains used: C-rfe-, SH3862 HfrH14; C-rfe+, SH1605 Hfr- H14; and B-rfe+, SA464 HfrK1-2 (16). selecting recombinants with the donor ilv+ and mete+ alleles and the recipient strr and hisalleles. Since ilv and mete are very close to each other, most of these recombinants were expected to have inherited the donor C-rfe- allele situated between ilv and mete. Because rfewas not known to have any phenotypic effect in group B strains, this could not be ascertained directly. The presence of C-rfe- could, however, be identified indirectly by testing its effect on the expression of C-rfb+. This was done with one such C-ilv+ C-metE+ recombinant (SH4145) which was taken at random and crossed, as recipient, with a S. montevideo donor (S-6,7) (HfrH14 strain SH1605), selecting recombinants with the donor his+ and recipient strr alleles. Out of 20 such recombinants 6 were, like the recipient, S-4,12, whereas 14 were R of type Ra. The latter 14 recombinants were presumed to have inherited the donor C-rfb+ genes along with his; the fact that they were R indicated the presence of C-rfeinstead of B-rfe+ in them and, consequently, in their parent SH4145. SH4145 was found to be CA-. In another cross between the same original parents (donor SH3862 of group Cl with C-rfe-, recipient SH380 of group B, ilv- mete- hisstrr), we selected two recombinants that had mete+ from the group Cl parent but ilv - from the recipient, and tested their rfe character by the same backcross method. One gave his+ S-4, 12 and R recombinants and was, therefore, believed to have inherited the donor C-rfe-; the other one gave his+ S-4,12 and S-6,7 recombinants and was believed to have retained the recipient B-rfe+. Of these, the former was CAand the latter was CA+, as expected if C-rfe- is unable to support CA synthesis in group B strains. The close association of the CA- character with C-rfe- was demonstrated further by analyzing 16 recombinants with the donor ilv+ allele from each cross of SH3862 (HrfH14, group C1, with C-rfe-) or SH1605 (HfrH14, group Cl, with C-rfe+) with an ilv- S. typhimurium recipient, SH3879. In the first cross, 15 of the recombinants were CA-; the known linkage of rfe with ilv is greater than 90%, indicating that the 15/16 figure corresponds very nicely with that expected if C-rfe- determines a CA- phenotype. In the latter cross all ilv + recombinants were CA+, the phenotype expected of strains with B-rfe+ or C-rfe+. From the CA- strain SH4145 (C-rfe-, B-rfb+; type 10 in Table 3), we then selected, after diethylsulfate treatment, an ilv- mutant and crossed it with rfe+ donors to select recombi-

5 VOL. 119, 1974 COMMON ANTIGEN IN SALMONELLA GROUPS B A&D C1 769 nants that had inherited donor rfe+ ilv+ alleles. When the rfe+ donor was of group Cl (HfrH14 strain SH1605), strains of type 11 (Table 3) were produced. They were found to be CA+; the rfe+ character was confirmed by crossing them with an S. montevideo S-6,7 donor and selecting recombinants with the donor his+ allele (either S-6,7 [majority] or S-4,12). If the rfe+ donor was a S. typhimurium strain (e.g., SA464, HfrKl-2), strains of type 12 (Table 3) were produced and found to be CA+. (The type 12 strains resemble wild-type group B strains in respect of both rfe and rfb and were, therefore, expected to be CA+.) The rfe+ character of these strains was confirmed by a cross with an S. montevideo S-6,7 donor; his+ recombinants were again S-4, 12 or S-6, 7. In the bottom half of Table 3 we have the corresponding derivatives of group Cl, all with C-rfb+. First, strain type 9, the C-rfe- mutant shown in Table 2, is R and CA- because of the rfe mutation. This strain was crossed with rfe+ donors of either group Cl (HfrH14 strain SH1605) or group B (HfrK1-2 strain SA464), the same donors used above. Recombinants with the donor ilv+ allele were selected. Those that had become S-6,7 were considered to have inherited the donor rfe+ capable of permitting the expression of the recipient C-rfb+; they always represented approximately 90% of the ilv+ recombinants. Of these rfe+ recombinants, only those with C-rfe+ (from the group Cl parent SH1605), strain type 13 in Table 3, are CA+. Strains of type 14, with B-rfe+ from their group B donor and therefore also capable of making an S-6, 7-type LPS, have only trace amounts of CA activity. We can say, therefore, that B-rfe+ contains some genes that, together with B-rfb+, are both necessary and enough to support the synthesis of CA (strain 12 compared with 10). B-rfe+ and C-rfb+ support the synthesis of 0-6,7 but support that of CA (strain 14) defectively. C-rfe+ suffices for CA synthesis irrespective of rfb. DISCUSSION The data here, together with our previous work with group L strains (9), demonstrate that the rfe genes are required for the synthesis of CA, not only in groups L and Cl but also in group B in which they are not needed in LPS synthesis. The primary product of the rfe gene(s) is not known at all, but the association with the biosynthesis of both CA and certain types of LPS should contribute towards a working hypothesis, hampered, it is true, by our present lack of knowledge of the chemical nature of CA. In fact, circumstantial evidence suggests that the rfe product may be connected with the synthesis of a carrier molecule for the assembly of 0 polysaccharides of types 6,7 (group Cl), 21 (group L), and possibly others (no 0 hapten in rfe mutants, yet their rfb genes suffice to determine the structure of the 0 polysaccharide of types 6, 7 or 21, respectively, when transferred into a group B recipient strain). This would necessitate a different sort of carrier being used for the 6,7-type side chain than the ACL used for 0 side chain synthesis and glucosylation in groups B and E (12, 17). This is not unfeasible; in fact, there is preliminary evidence suggesting that the 0-6, 7-hapten is built differently from the 0-4,12-hapten (J. Gmeiner and H. Nikaido, personal communication). The postulated second carrier could still be related to the known C55-isoprenol ACL, which appears to be employed for the synthesis of very many cell wall polysaccharides (12); it could, for example, be this ACL modified by the rfe product. The second carrier would then be used to synthesize rfe-dependent structures: the 0 side chains of Salmonella groups C1 and L, the Ti side chain (manuscript in preparation) found in the LPS of many salmonellae, and the CA found in all enteric bacteria. This hypothesis suggests very strongly that the CA contains a sugar moiety. It also suggests that a "rfe+" gene may be found in all enteric bacteria irrespective of whether the 0 antigen requires its function. Along this line, it explains the originally surprising finding that group B strains have a rfe+ allele capable of supporting the synthesis of the 0-6,7 polysaccharide. Although all C-rfe+ strains are CA+ irrespective of the quality of their rfb region, the B-rfb genes appear to have a role in the synthesis of CA in strains with B-rfe+ (strain types 3, 4, 5 in Table 1; 14 in Table 3). In all these strains, however, a weak CA activity was recovered. This suggests that the B-rfe+ product may be, even in these strains, qualitatively sufficient to support the synthesis of CA, but its amount is very much reduced. As a possible reason for this reduction H. Nikaido (University of California, Berkeley, Calif.) suggested that much of the "second carrier" may be consumed in 0-6,7 synthesis, leaving little for the pathway of CA synthesis (whereas the B-rfb+ product does not trap this carrier). Strains 4 and 5 of Table 1 (B-rfe+, C-rfb-, mutated or otherwise rendered nonfunctional), however, do not make 0-6,7 polysaccharide, but they could still make an

6 770 MAKELA AND MAYER J. BAcTEOL. abortive start of 0 side chain synthesis sufficient to sequester the carrier away from the available pool. Another possibility with respect to the CAtraec phenotype in strains of this type (B-rfe+, C-rfb+ or C-rfb) is that their apparent CA reactivity is, in fact, a cross-reaction between the anti-ca serum and a CA-related molecule (e.g., a precursor of CA) coating the red cells. This sort of CA-related molecule would then not be produced in rfe- bacteria which are CA-. We feel that further speculation is not useful at this stage because a more definitive answer could be qhteinn b,y stu4. su.tips With 44ktipus in the rfb giqn (13X, and ouch a study in progress. ACKNOWLEDGMENTS We thank Marianne Hovi and Marjukka Brandt for excellent assistance. The work in Helsinki was supported by grants from the Finnish Medical Research Council, the Sigrid Juselius Foundation, and Academia Scientiarum Fennica. LITERATURE CITE) 1. Edwards, P. R., and D. W. Bruner Serological identification of Salmonella cultures. Circular. Kentucky Agricultural Experiment Station Hammarstrom, S., H. E. Carlson, P. Perlmann, and S. Svensson Immunochemistry of the common antigen of Enterobacteriaceae (Kunin). Relation to lipopolysaccharide core structure. J. Exp. Med. 134: Johns, M. A., R. E. Whiteside, E. E. Baker, and W. R. McCabe Common enterobacterial antigen. I. Isolation and purification from Salmonella typhosa 0:901. J. Immunol. 110: Kauffmann, F The bacteriology of Enterobacteriaceae. Munksgaard, Copenhagen. 5. Kunin, C. M., and M. V. Beard Serologic studies of O antigens of Escherichia coli by means of the hemagglutination test. J. Bacteriol. 85:541-M Kunin, C. M., M. V. Beard, and N. E. Halmagyi Evidence for a common hapten associated with endotoxin fractions of E. coli and other Enterobacteriaceae. Proc. Soc. Exp. Biol. Med. 111: MMkela, P. H Genetic determination of the 0 antigens of Salmonella groups B (4, 5, 12) and C, (6, 7) J. Bacteriol. 91: Milkell, P. H., M. Jahkola, and 0. Lfideritz A new gene cluster rfe concerned with the biosynthesis of Salmonella lipopolysaccharide. J. Gen. Microbiol. 60: Mikela, P. H., H. Mayer, H. Y. Whang, and E. Neter Participation of lipopolysaccharide genes in the determination of the enterobacterial common antigen: analysis of R mutants of Salmonella minnesota. J. Bacteriol. 119: Mikeli, P. H., and B. A. D. Stocker Genetics of polysaccharide biosynthesis. Annu. Rev. Genet. 3: Mayer, H., and G. Schmidt Hlmagglutinine gegen ein gemeinsames Enterobacterioceen-Antigen in E. coli RI-Antiseren. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. Abt. 1 Orig. 216: Nikaido, H Biosynthesis of cell wall lipopolysaccharide in Gram-negative enteric bacteria. Advan. Enzymol. 31: Nikaido, H., M. Levinthal, K. Nikaido, and K. Nakane Extended deletions in the histidine-rough-b region of the salmonella chromosome. Proc. Nat. Acad. Sci. U.S.A. 57: Nikaido, H., K. Nikaido, and P. H. Mikela Genetic determination of enzymes synthesizing 0- specific sugars of Salmonella lipopolysaccharide. J. Bacteriol. 91: Sanderson, K. E Linkage map of Salmonella typhimurium, edition IV. Bacteriol. Rev. 36: Sanderson, K. E., H. Ross, L. Ziegler, and P. H. Mikela F+, Hfr, and F' strains of Salmonella typhimurium and Salmonella abony. Bacteriol. Rev. 36: Stocker, B. A. D., and P. H. Mikeli Genetic aspects of biosynthesis and structure of Salmonella lipopolysaccharide, p In G. Weinbaum, S. Kadis, and S. J. Ajl (ed.), Microbial toxins, vol. 4. Academic Press Inc., New York. 18. Whang, H. Y., H. Mayer, G. Schmidt, and E. Neter Immunogenicity of the common enterobacterial antigen produced by smooth and rough strains. Infect. Immunity 6: Wilkinson, R. G., P. Gemaki, and B. A. D. Stocker Non-smooth mutants of Salmonella typhimurium: differentiation by phage sensitivity and genetic mapping. J. Gen. Microbiol. 70: