Vol. 13, No. 6 Printed in U.SA.
INFCTON AND IMMUNITY, June 1976, p. 1647-1653 Copyright C 1976 American Society for Microbiology
T2 Lipopolysaccharide Antigen of Salmonella: Genetic Determination of T2 and Properties of the T2, T2,S, and T2,SR Forms V. V. VALTONEN, MATTI SARVAS, AND P. HELENA MAKELA* Central Public Health Laboratory, State Serum Institute,* and Department of Serology and Bacteriology, University of Helsinki; Helsinki, Finland
Received for publication 26 December 1975
The T2 antigenic form of Salmonella bareilly was examined. The absence of 0 specificity in this strain was shown to be due to its nonfunctional rfb genes; when the rfb gene cluster was replaced by the rfb cluster derived from smooth donor strains, T2,S and T2,SR recombinants were produced that expressed both T2 and either 0-6,7 or 0-4,12 specificity, depending on 0 antigen of the donor strain. The T2, T2,S, and T2,SR forms were all unstable on culture and segregated T2-negative forms (R, S, and SR, respectively) at a high rate. In all these respects the T2 antigen closely resembled the other T-form antigen, Ti. The genes responsible for the T2 antigen, rfu, were not close to rtb, but their precise location and relation to rft (which determines Ti antigen) could not be discovered because of the instability of the T2 form and low recombination frequency in the necessary interspecies crosses.
The lipopolysaccharide (LPS) in the cell walls of Salmonellae affects not only their growth characteristics, serological specificity, and phage sensitivity, but also their pathogenicity (12, 20). The normal, smooth (S) form has long 0-specific side chains in its LPS; if these are lost (as is the case in the commonly seen rough [R] forms), all these parameters are changed. Sometimes a third, so-called T form is found among strains from natural sources (3) or is produced in the laboratory (19). The T forms were described by Kauffmann as transient forms because they are culturally smooth but give R variants at high rate. The T forms have their own antigenic specificity, unrelated to any 0 antigen, and they are probably of intermediate virulence between S and R (13, 21). In the most common T form, Ti, the serological specificity is based on Ti side chains in the LPS side chains that are long polymers of galacto- and ribofuranosides, attached to the LPS core in the same way as are 0 side chains in the S form (1) (Fig. 1). The genetic determinants of the Ti side chains reside in an rft locus or cluster of loci (16) (see map in Fig. 2), whereas the 0 side chains are determined by genes at the separate rib cluster (10). The complete LPS core, whose synthesis requires the function of the rfa genes, is necessary as the attachment site for both the Ti and 0 side chains (10, 16). Strains that have both functional rft+ and functional rfb+ genes have been prepared in the
laboratory. In them, both Ti and 0 side chains are formed and transferred to the core (16, 17). The resulting T1,S form has both serological specificities, but the amount of 0 side chain material is less than in the corresponding S form (6, 16). The biosynthesis of the structurally exceptional Ti side chain, whose monosaccharide components are in the furanosidic form, is being studied (11, 18). Besides this Ti form, two other T forms have been described, called T2 and T3. T2 has been found in nature as a derivative of Salmonella bareilly, of group C0 (4), while T3 was identified tentatively in S. upphill, of 0 group T (3). Both are apparently rare and have only been, to our knowledge, found this once. In the present work we have studied the genetic basis of T2, specially whether analogies could be drawn to the determinants of the Ti or 0 side chains. The chemical basis of the T2 specificity has been concurrently analyzed, partly by using mutants and recombinants described in this paper (2). The T2 determinant appears to be an N-acylglucosamine, with an unknown substituent at C3 or C4, attached to the LPS core in the same way as are 0 or Ti side chains. MATERIALS AND METHODS Bacterial strains (Table 1). The S. bareilly T2 strains are sublines of the T2 strain described by Kauffhnann (4). One, SH 144, was received from B. A. D. Stocker, Stanford University, Stanford, Calif.
1647
1648
INFECT. IMMUN.
VALTONEN, SARVAS, AND MAKELA GNAc
Gal II
1 II-Gal I-Glc I-(Hep, KDO, etc. -lipid A
12 Z--4 Glc
core
Z = specific side chain, which is in R form = none in T2 form = substituted GNAcyl in Ti form = (Galf)n, (Ribf)m in S(6,7) form = (Glc,, Man4, GNAc,). Abe in S(4,12) form = (-Man-Rha-Gal-)n Abe in SR(4,12) form = Man Rha-Gal FIG. 1. Schematic structure of the lipopolysaccharide of various Salmonella forms (1, 2, 7). Abbreviations: Glc, D-glucose; Gal, D-galactose; GNAc, N-acetylglucosamine; GNAcyl, N-acylglucosamine; Hep, heptose; KDO, ketodeoxyoctonate; Rib, D-ribose; Man, D-mannose; Abe, abequose; Rha, L-rhamnose. All sugars are pyranoses except those with an "f' suffix, which are furanoses.
(
FIG. 2. Simplified map of the Salmonella chroshowing the position of the genes and the characteristics of the donor (Hfr and F') strains used (14, 15).
inoculated previously with the appropriate bacteria in a soft-agar layer. Serological tests. Overnight bacterial growth from nutrient agar plates was used for agglutination. The tests were done on glass slides using 4% NaCl or antisera diluted appropriately in 0.2% NaCl containing 0.5% phenol as preservative. The antisera were prepared in rabbits as described by Kauffmann (5); the immunogen for anti-T2 serum was a heated (2 h at 100 C) suspension of SH 144. Crosses. Conjugation was the only method of genetic analysis used (15). Equal amounts of donor (Hfr or F') and recipient broth culture were mixed, and the mixture was incubated for 2 h without shaking. Aliquots (0.1 ml) of the mixture were then plated onto appropriate selective media. The recombinants appearing after 48 h of incubation were restreaked on nutrient agar plates, and single colonies from these were used for further tests. Culture media. Davis minimal medium (15) was used as selective medium with added agar (1.2%), glucose (0.2%), required amino acids (20 ,ug/ml), and/or streptomycin (1 mg/ml).
mosome
The second T2 line, SF 1187, was obtained from 0. Luderitz, Max-Planck-Institut fur Immunbiologie, Freiburg/Breisgau, Germany. Both strains were identical in agglutination tests and in their sensitivity to a range of S- or R-specific phages (see below). Mutants were selected from these strains after diethylsulfate treatment (9). Known Hfr and F' strains of Salmonella species were used as genetic donors (15). Phages. The rough-specific phage stocks were obtained from B. A. D. Stocker and propagated here; their properties have been described (24). When the sensitivity of a bacterial strain to phages was determined, phage lysates were applied as drops of about 108 plaque-forming units on a nutrient agar plate
RESULTS Properties of T2 strains. (i) Stability. When an overnight broth culture of a T2 strain was plated on nutrient agar, several of the developing colonies were morphologically rough, the rest appearing smooth. The agglutination reactions of these colony types confirmed them to be R and T2, respectively (Table 2). The R form was stable on subculture; the T2 form continued to segregate R colonies. The frequency of R forms in the overnight broth culture of T2 forms was approximately 5% and thus higher than in cultures of Ti strains. The formation of the R forms seemed to be a mutation from T2 positive to T2 negative. The same loss of the T2 antigen was later noticed in T2,S and T2,SR forms;
VOL. 13, 1976
1649
T2 LPS ANTIGEN OF SALMONELLA
TABLE 1. Salmonella strains used Strain no.
0
Species
C, C, C,
SF 1187 SH 144 SH 713 SH 1308 SH 1844 SH 1914 SH 2033 SH 3416 SH 5121
S. bareilly S. bareilly S. montevideo S. paratyphi B S. bareilly S. bareilly S. bareilly S. abony S. abony
SH 5152
S. abony hybrid B
B
C,
C, C, B B
Marker genesb
Mating type
Serotype
group'
Reference or parent strain
(4) (4) (8)
FFHfrH14 aro-852 leu-1357 met-1183 ilv-1123 FS59 Fhis-6143 Fhis-6143 gal-928 his-6143 FT1,S(1,4,5,12) HfrH1 met-1151 aro-851 str-501 HfrH2 met-1151 aro-851 S(1,4,5,12) his-6163 str-501 HfrH2 met-1151 aro-851 str-501 S(6,7) T2 HT2 S(6,7) Ti T2 R T2
(16) SF 1187 SH 1844 SH 1844 (15) SW 1403 from SH 713 x SH 5121
HfrH2 met-1151 aro-851 str-501 (15) B SW 1403 S. abony S(1,4,5,12) a The characteristic 0 antigens for group B are (1),4,(5),12; those for group C, are 6,7. b Standard gene symbols used (14). TABLE 2. Serological reactions and phage sensitivity of several T2+ and T2- forms of Salmonella bareilly compared with certain standard strains Sensitivity to phagesO Slide agglutination in:a Strain
4% AntiNaCi R
S. bareilly T2 R
Tic
~
++ -
S. typhimu-
T2
+
++
++ +
++
T2,S(6,7)r
S(6,7)r T2,SR(4,12) SR(4,12Y
Anti-
Anti- AntiTi 4,12
_
++ ++
Anti-
P22 FO 6SR
6,7P2FO6R
Br60 B6
Ffm Ffn
B
P221 2
X174
r
21C1OX7
+
+
+or-
+or-
-
-
+
+
+
+
+
-
-
+
+
+
-
-
+
+
+ or -
+
-
-
+
++
-
++
-
+
-
-
-
-
-
-
++
-
+
--
-
-
-
-
_ _
_ -
+ +
_ _
_ _
_ _
_ _
_ _
_ _
_ _
_
+
+
+
+
+
+
-
+
-
-
-
-
-
-
±
+ +
++ _-
_
+ +
++
++
++
++
+
riUMd
R(rfbi)
Tl
SR(4,12) S.
montevideo
+
-
++
-
-
-
+
+
+
+
+
+
-
+
±
+
-
-
+
-
-
+
-
-
-
-
+
-
-
++
++
++
+
+
_
+
+
+
+
+
-
-
ND ND ND
R (rfl) Tl
-
+
S(6,7)
-
-
++
-
-
|
-
-
-
+
+
+
+
+
-
-
++
-
+
-
-
-
-
-
-
a Antisera appropriately diluted in 0.2% NaCl. Symbols: + +, Strong agglutination with large clumps; +, strong agglutination, small clumps; ±, weak agglutination; -, no agglutination. b Phages applied as drops of 106 to 107 phages on nutrient agar plates previously spread with 0.1 ml of an overnight broth culture of the bacteria; incubated at 37 C. ND, Not done. Strains obtained as recombinants in crosses reported in this paper. d Several strains of each type described earlier (9, 16, 17). c
these mutated to S and SR, respectively (see below). (ii) Colony morphology. The T2-positive forms, either T2, T2,S, or T2,SR, were more opalescent than their Ti-negative mutants. This difference was more pronounced after longer incubation and at low temperature (20 C). The colony morphology and its dependence on temperature was reminiscent of the behavior of mucoid mutants. To exclude the possibility that the T2 forms were misinterpreted mucoid mutants, we isolated a set of true mucoid mutants and compared them with T2; they proved different both in their cultural
properties and the chemistry of the cell wall (M. Sarvas, M. Malinen, M. Nurminen, and P. H. Makel, submitted for publication). (iii) Agglutination reactions. Suspensions of the T2 forn were stable in 4% NaCl and agglutinated specifically, with formation of large clumps in anti-T2 serum (Table 2). In addition, the T2 form agglutinated to some extent in anti-R sera. For practical purposes the saline reaction was sufficient to differentiate T2 colonies from R colonies, which clumped strongly in saline and in anti-R, anti-T2, and anti-Ti sera and also to some extent in various (unabsorbed) anti-O sera. The strong reaction between R
1650
VALTONEN, SARVAS, AND MAKELA
colonies and anti-T2 sera may indicate the presence of anti-R antibodies in these sera, caused by the R mutants unavoidably present in the T2 batch used for immunization. Alternatively, this reaction may represent a cross-reaction between the R and T2 structures, but this could not be shown conclusively. (The same situation holds for the relation of R and Ti.) The T2,S(6,7) form found among recombinants (see below) agglutinated strongly and specifically in both anti-T2 and anti-0(6,7) sera, but not in saline, whereas its S(6,7) mutants agglutinated in anti-0(6,7) sera only. The T2,SR(4,12) form agglutinated strongly in anti0(4,12) sera, weakly in anti-R sera, and to a very slight degree in saline, whereas its SR(4,12) mutants agglutinated in anti-0(4,12) but not in anti-T2 sera and gave a slightly stronger reaction in anti-R sera and saline. A chemical examination of the LPS extracted from the various serologically classified forms confirmed the classification (2) into the various T2-positive and T2-negative forms. (iv) Phage sensitivity. When tested with a set of S- or R-specific phages (Table 2), the T2 form showed a sensitivity pattern closely resembling that known for R mutants with a complete LPS core (24). The R-specific phage P221 did not give plaques on the T2 strain, but since this phage did not grow on R mutants of the T2 strain either, it is assumed that this insensitivity was due to the fact that the T2 strains were derivatives of S. bareilly (O group Cl) rather than S. typhimurium (O group B), in which the phage pattems were originally determined (24). A similar nonsensitivity to some Rspecific phages has been noticed in R mutants of Salmonella groups Cl and L (9). In addition, the T2 form was completely resistant to phage Br2 and relatively resistant to the R-specific phage Ffm, whereas its R mutants were sensitive to both of them. It seems possible that the T2-specific structures prevent the access of these phages to their receptor sites in the LPS core while allowing some other Rspecific phages to attach. The phage sensitivity of the T2,S(6,7) and T2,SR(4,12) forms was similar to that of their T2-negative mutants. The SR(4,12) form was sensitive to phage FO only; although similar forms of Salmonella typhimurium are sensitive to both FO and P221, this S. bareilly derivative was not expected to be P221 sensitive (see above). Production of T2 derivatives with 0 specificity. Two main alternatives could account for the lack of 0 specificity in the T2 form. The first assumes that the genetic determinants of the T2 structure, called here rfu, are allelic to the
INFECT. IMMUN.
rtb cluster and then bacteria make either the rfu-directed T2 structure of the rtb-directed 0 side chain. The second assumes that rfu and rtb genes are independent of each other and the lack of 0 specificity in the T2 form is due to mutations of the rtb genes. This latter is the case in Ti forms in which rft genes determine Ti specificity (16). To decide between these alternatives, we replaced the rtb region of the T2 form with a functional rib from another, S strain; if recombinants with both T2 and 0-specific structures are found, the first alternative is excluded. This genetic exchange can be done by crossing a his- mutant of the T2 strain with a smooth donor strain and selecting recombinants that had received the donor his+ allele. Since the rib cluster is close to his, many his+ recombinants also receive the donor rib (8). When the S(1,4,5,12) HfrH2 donor SW 1403 (S. abony, 0 group B; see Fig. 2) was in this way crossed with the T2 his- recipient SH 1844 (crosses A and B, Table 3), about 40% of the his+ recombinants showed the donor 0 specificity 4,12, as expected (0 factors 1 and 5, also present in the donor strain but not determined by rfb genes, were not tested). These recombinants also agglutinated in anti-R sera and to a slight degree in saline in a way characteristic of SR forms. This is the form expected in crosses of group B donors with group C, recipients (8) in which the recombinants receive the rib genes of the donor but not the remote rfc gene(s) that would be required for the polymerization of the donor-type 0-specific units. Most of these SR(4,12) recombinants were also agglutinated in the anti-T2 serum, indicating that they had retained the rfu genes determining T2 specificity. In cross B, three out of the 18 recombinants with 0-4,12 appeared to have lost the T2 specificity. It was not possible to find outAvhether they had lost their rfu+ genes in the recombination process or were the result of a mutation from T2 positive to T2 negative (in the recipient or the recombinant). In either case, the results show that rfu is not allelic or even close to the rib. The lack of 0 antigen in the T2 form must hence be due to an rtb defect(s). For comparison, a T2-negative mutant of the T2 recipient parent in crosses A and B was used as recipient in a similar cross with the same donor (cross C, Table 3). Now 30% of the 35 his+ recombinants had the donor 0 specificity; although most of these recombinants were SR(4,12), one was S(4,12), indicating that it had received both the rtb and rfc genes from the donor. No T2-positive forms were obtained. Because S. bareilly is of group C,, characterized by the 0 specificity 6,7, we also wanted to
T2 LPS ANTIGEN OF SALMONELLA
VOL. 13, 1976
1651
TABLE 3. Analysis ofthe his+ recombinants in crosses between a his+ met- S(4,12) Salmonella abony donor SW 1403 and his- met' S. bareilly recipients, either T2 or R Cross
Serotype
Donor S. abony SW 1403 Recipient S. bareilly SH 1844 his+ recombinants: 52 in cross A l 42 in cross B
S(4,12) T2 T2 T2,SR(4,12) SR(4,12)
Genes involved in the crosses"
Percent ofeach recombinant class cross A cross B
hibs +
rfb +(4,12)
D D D
r D D
d
60 40 0
57 36 7
rfu' + r r D or r'
rfc +
metP
_C
+ r r r
ND r r
+ _ _ _ R r ND r D 71" R r r D D 26" SR(4,12) r D D D 39 S(4,12) a Abbreviations: D, Donor allele; r, recipient allele for each gene locus or cluster tested; ND, not determined. b Markers used for the selection of recombinant. rfu stands here for any gene required for the production of the T2 antigen. d Inferred from the outcome of the cross. " rfc+ together with rfb+ (4,12) leads to an S(4,12) serotype, whereas rfc- with rfb+ (4,12) leads to SR(4,12). The rfc character cannot be assessed with rtb- or rfb+ (6,7), but was deduced from the behavior of the recombinants produced. f rfu- can be a donor character or a recipient rfu+ that had mutated to rfu-. D Results for cross C.
Recipient S. bareilly SH 1914 his+ recombinants 35 in cross C
"
check whether the T2 strain could make the 06,7 antigen if given complete rib genes of this 0 type. The yield of recombinants with an 0-6,7 HfrH14 donor, SH 713 of group C,, was very low, however. An 0-6,7 derivative SH 5152 of the HfrH2 strain used in crosses A and B was a better donor, probably because it transfers his and rib efficiently close to its origin. Among 38 his+ recombinants isolated (SH 5152 x SH 1844), 22 were T2, nine were T2,S(6,7), four were S(6,7), and three were R. It seems probable that the S and R recombinants were derived from T2-negative mutants of the recipient. The production of T2,S(6,7) recombinants showed, however, that the T2 strain was capable of synthesizing the 0-6,7-type side chain when it was provided with functional rib genes of this type. Attempts to investigate the relation of Ti and T2 determinants and to localize the rfu gene(s). We crossed a Ti-positive S. paratyphi B strain SH 1308 (rft+ rfb+ ilv- met-) containing the episome FS59 as donor to the T2-positive strain SH 2033 (rfu+ gal- his- rib-) as recipient, and selected the gal+ ilv+ met+ recombinants. The frequency of recombinants was low, approximately 3 x 10-7. Of 90 recombinants obtained in two separate crosses, 88 agglutinated like T2, and two agglutinated like Ti. No recombinants with both T2 and Ti specificity were found. In further crosses the donor was SH 3416, a Ti-positive derivative of S. abony HfrHl; the recipient was the same SH 2033. Of 158 gal+ recombinants, two were Ti, nine were R, and 147 were T2. No strains with
both Ti and T2 were seen. The Ti-positive recombinants probably had received the donor rft+ genes; their frequency (4/248) was very much lower than expected on the basis of the previously observed approximately 50%o linkage between rft and gal. We believe that this poor linkage is due to low homology between the parents in these crosses
(15). The absence of T1,T2 recombinants might suggest that Ti and T2 are determined by allelic genes. It is also possible that either Ti or T2 is epistatic to the other. The small number of Ti-positive recombinants reduces the value of these crosses; it is possible that recombinants were preferentially formed from T-negative mutants of either donor or recipient (or both) so that no recombinants would be expected to have rft+ and rfu+ The poor recombination frequency, low linkage, and frequency of T2-negative mutants in the T2 strains proved prohibitive to attempts at localizing the rfu genes. More conclusive results would be expected from crosses using T2positive donors, but all our attempts at producing such were unsuccessful, e.g., all recombinants from the above-mentioned crosses between F' donors and T2 recipients were F-. DISCUSSION The Ti form has been previously shown to be genetically rft+ and ri- and to mutate at high frequency to the Tl-negative form rft- rib-. (We have chosen to use rft+ and rfu + as the
1652
VALTONEN, SARVAS, AND MAKELA
symbols for the uncommon form of postulated genes determining the uncommon antigens Ti and T2 and the corresponding symbols with superscript "minus" for the common form, not determining any such antigen. In this way "plus" is connected with a functional form and "minus" is connected with a nonfunctional form; note that this is opposite of the usage specified in the standard convention of bacterial genetics.) The rib- of the Ti form could be replaced by a functional rfb gene cluster to produce T1,S or T1,SR forms containing both Ti and 0 side chains in their LPS and showing both Ti and 0 serological specificity (16, 17). Furthermore, both the Ti and 0 side chains have been shown to be attached to the LPS core in the same position, carbon 4 of the terminal glucose, which carries N-acetylglucosamine at carbon 2 (1). The T2 form seems to be analogous to Ti in all these respects. The T2 form isolated from a natural source is genetically rfu+ rfb- and mutates to R (rfu-). By genetic manipulation, T2,S and T2,SR forms (rfu+ rfb+) can be derived from it; these can in their turn mutate to rfu- at the same high rate as T2 mutates to R. The T2 chemical determinant (a substituted N-acylglucosamine) is linked to carbon 4 of the glucose II of the LPS core in the same way as are 0 and Ti side chains (2) (Fig. 1). In a T2,SR form, the amount of 0-specific material was reduced compared with its T2-negative (that is, SR) mutant, suggesting competition between the T2 and 0 determinants (2). This outcome is expected since they share the same attachment site. The Ti determinant is a long polysaccharide chain, whereas T2 specificity is based on a substituted monosaccharide only. The long Ti side chains are apparently relatively few in number; consequently they do not "cover up" the rest of the LPS very efficiently, and the Ti form is nearly as sensitive to the R-specific phages as is its Ti-negative mutant. The T2 determinant occupies a larger fraction of the core stubs. In the study of Bruneteau et al. (2), more than 50% of core stubs in T2 form LPS had the T2 determinant N-acylglucosamine; the proportion could not be determined exactly because of the variable occurrence of R mutants in the culture batches from which the LPS was extracted. In the same study the T2,SR form showed a ratio of 0 units to core to be 1:2 (calculated from a 1:4 ratio of rhamnose to glucose) in a batch in which we determined the frequency of T2-negative SR mutants to be exceptionally low, approximatively 5%. As the 0 unit-to-core ratio in the corresponding SR form was 1:1, this would indicate that 50%o of the core
INFECT. IMMUN.
stubs were substituted by the T2 unit. In this paper (Table 2) we saw that the presence of T2 determinants reduces the sensitivity of the T2 form to several R-specific phages compared with its R mutants. An analogous situation has been seen in SR forms in which every core stub is covered by a trisaccharide 0 unit and which are resistant to most of the R-phages (24). ACKNOWLEDGMENTS We thank Seijasisko Suurmnkki for skillful technical assistance. This research was supported by Public Health Service grant GM-12046 from the National Institute of General Medical Sciences and by grants from the Finnish Medical Research council and Sigrid Juselius Foundation. LITERATURE CITED 1. Berst, M., C. G. Hellerqvist, B. Lindberg, 0. Luderitz, S. Svensson, and 0. Westphal. 1969. Structural investigation on TI lipopolysaccharides. Eur. J. Biochem. 11:353-359. 2. Bruneteau, M., W. A. Volk, P. P. Singh, and 0. Luderitz. 1974. Structural investigations on theSalmonella T2 lipopolysaccharide. Eur. J. Biochem. 43:501-508. 3. Kauffmann, F. 1956. A new antigen of Salmonella paratyphi B and Salmonella typhimurium. Acta Pathol. Microbiol. Scand. 39:299-304. 4. Kauffmann, F. 1957. On the T antigen of Salmonella bareilly. Acta Pathol. Microbiol. Scand. 40:343-344. 5. Kauffmann, F. 1966. The bacteriology of enterobacteriaceae. Munksgaard, Copenhagen. 6. Lindberg, A. A., M. Sa.rvas, and P. H. Makela. 1970. Bacteriophage attachment to the somatic antigen of Salmonella: effect of 0-specific structures in leaky R mutants and S, Ti hybrids. Infect. Immun. 1:88-97. 7. Ltideritz, O., 0. Westphal, A. M. Staub, and H. Nikaido. 1971. Isolation and chemical and immunological characterization of bacterial lipopolysaccharides, p. 145-234 In G. Weinbaum, S. Kadis, and A. J. Ajl (ed.), Microbial toxins, vol. IV. Academic Press Inc., New York. 8. Mrikela, P. H. 1966. Genetic determination of the 0 antigens of Salmonella groups B (4,5,12) and C, (6,7). J. Bacteriol. 91:1115-1125. 9. Mikela, P. H., M. Jahkola, and 0. Ltideritz. 1970. A new gene cluster rfe concerned with the biosynthesis of Salmonella lipopolysaccharide. J. Gen. Microbiol. 60:91-106. 10. Mikela, P. H., and B. A. D. Stocker. 1969. Genetics of polysaccharide biosynthesis. Annu. Rev. Genet. 3:291-322. 11. Nikaido, H., and M. Sarvas. 1971. Biosynthesis of the Ti antigen in Salmonella: Biosynthesis on a cell-free system. J. Bacteriol. 105:1073-1082. 12. Roantree, R. J. 1971. The relationship of lipopolysaccharide structure to bacterial virulence, p. 1-38 In G. Weinbaum, S. Kadis, and A. J. Ajl (ed.), Microbial toxins, vol. IV. Academic Press Inc., New York. 13. Ruschman, E., and 0. Luderitz. 1971. The virulence for mice of Salmonella typhimurium Ti mutants. Zentralbl. Bakteriol. Parasitenkd. Infektronskr. Hyg. I Abt. Orig. 216:185-192. 14. Sanderson, K. E. 1972. The linkage map of Salmonella typhimurium. Bacteriol. Rev. 36:558-586. 15. Sanderson, K. E., H. Ross, L. Ziegler, and P. H. Mikelii. 1972. F+, Hfr, and F' strains of Salmonella
VOL. 13, 1976
typhimurium and Salmonella abony. Bacteriol. Rev. 36:608-637. 16. Sarvas, M. 1967. Inheritance of Salmonella Ti antigen. Ann. Med. Exp. Fenn. 45:447-471. 17. Sarvas, M., and P. H. Makelai. 1965. The production, by recombination, of Salmonella forms with both Ti and 0-specificities. Acta Pathol. Microbiol. Scand. 65:654-656. 18. Sarvas, M. and H. Nikaido. 1971. Biosynthesis of Ti antigen in Salmonella: Origin of D-galactofuranose and D-ribofuranose residues. J. Bacteriol. 105:1063-
T2 LPS ANTIGEN OF SALMONELLA
aspects of biosynthesis and structure of Salmonella lipopolysaccharide, p. 369-438. In G. Weinbaum, S.
21. 22.
23.
1072. 19. Schlosshardt, J. 1960. Unterschungen uber die Entstehung von T-Antigenen in S-R-Formenwechsel bei Salmonellen. Zentralbl. Bakteriol. Parasitenkd. Infektronskr. Hyg. I Abt. Orig. 177:176-185. 20. Stocker, B. A. D., and P. H. M&kel&. 1971. Genetic
1653
24.
Kadis, and A. J. Ajl (ed.), Microbial toxins, vol. IV. Academic Press Inc., New York. Valtonen, V. V., M. Sarvas, and P. H. Makela. 1971. The effects of Ti antigen on the virulence of Salmonella strains for mice. J. Gen. Microbiol. 69:99-106. Watanabe, T., T. Arai, and T. Hattori. 1970. Effects of cell wall polysaccharide on the mating ability of Salmonella typhimurium. Nature (London) 225:70-71. Wiedemann, B. and G. S. Schmidt. 1971. Structure and recipient ability in E. coli mutants. Ann. N. Y. Acad. Sci. 182:123-125. Wilkinson, R. G., P. Gemski, and B. A. D. Stocker. 1972. Non-smooth mutants of Salmonella typhimurium: differentiation by phage sensitivity and genetic mapping. J. Gen. Microbiol. 70:527-554.
INFCTON AND IMMUNITY, June 1976, p. 1647-1653 Copyright C 1976 American Society for Microbiology
T2 Lipopolysaccharide Antigen of Salmonella: Genetic Determination of T2 and Properties of the T2, T2,S, and T2,SR Forms V. V. VALTONEN, MATTI SARVAS, AND P. HELENA MAKELA* Central Public Health Laboratory, State Serum Institute,* and Department of Serology and Bacteriology, University of Helsinki; Helsinki, Finland
Received for publication 26 December 1975
The T2 antigenic form of Salmonella bareilly was examined. The absence of 0 specificity in this strain was shown to be due to its nonfunctional rfb genes; when the rfb gene cluster was replaced by the rfb cluster derived from smooth donor strains, T2,S and T2,SR recombinants were produced that expressed both T2 and either 0-6,7 or 0-4,12 specificity, depending on 0 antigen of the donor strain. The T2, T2,S, and T2,SR forms were all unstable on culture and segregated T2-negative forms (R, S, and SR, respectively) at a high rate. In all these respects the T2 antigen closely resembled the other T-form antigen, Ti. The genes responsible for the T2 antigen, rfu, were not close to rtb, but their precise location and relation to rft (which determines Ti antigen) could not be discovered because of the instability of the T2 form and low recombination frequency in the necessary interspecies crosses.
The lipopolysaccharide (LPS) in the cell walls of Salmonellae affects not only their growth characteristics, serological specificity, and phage sensitivity, but also their pathogenicity (12, 20). The normal, smooth (S) form has long 0-specific side chains in its LPS; if these are lost (as is the case in the commonly seen rough [R] forms), all these parameters are changed. Sometimes a third, so-called T form is found among strains from natural sources (3) or is produced in the laboratory (19). The T forms were described by Kauffmann as transient forms because they are culturally smooth but give R variants at high rate. The T forms have their own antigenic specificity, unrelated to any 0 antigen, and they are probably of intermediate virulence between S and R (13, 21). In the most common T form, Ti, the serological specificity is based on Ti side chains in the LPS side chains that are long polymers of galacto- and ribofuranosides, attached to the LPS core in the same way as are 0 side chains in the S form (1) (Fig. 1). The genetic determinants of the Ti side chains reside in an rft locus or cluster of loci (16) (see map in Fig. 2), whereas the 0 side chains are determined by genes at the separate rib cluster (10). The complete LPS core, whose synthesis requires the function of the rfa genes, is necessary as the attachment site for both the Ti and 0 side chains (10, 16). Strains that have both functional rft+ and functional rfb+ genes have been prepared in the
laboratory. In them, both Ti and 0 side chains are formed and transferred to the core (16, 17). The resulting T1,S form has both serological specificities, but the amount of 0 side chain material is less than in the corresponding S form (6, 16). The biosynthesis of the structurally exceptional Ti side chain, whose monosaccharide components are in the furanosidic form, is being studied (11, 18). Besides this Ti form, two other T forms have been described, called T2 and T3. T2 has been found in nature as a derivative of Salmonella bareilly, of group C0 (4), while T3 was identified tentatively in S. upphill, of 0 group T (3). Both are apparently rare and have only been, to our knowledge, found this once. In the present work we have studied the genetic basis of T2, specially whether analogies could be drawn to the determinants of the Ti or 0 side chains. The chemical basis of the T2 specificity has been concurrently analyzed, partly by using mutants and recombinants described in this paper (2). The T2 determinant appears to be an N-acylglucosamine, with an unknown substituent at C3 or C4, attached to the LPS core in the same way as are 0 or Ti side chains. MATERIALS AND METHODS Bacterial strains (Table 1). The S. bareilly T2 strains are sublines of the T2 strain described by Kauffhnann (4). One, SH 144, was received from B. A. D. Stocker, Stanford University, Stanford, Calif.
1647
1648
INFECT. IMMUN.
VALTONEN, SARVAS, AND MAKELA GNAc
Gal II
1 II-Gal I-Glc I-(Hep, KDO, etc. -lipid A
12 Z--4 Glc
core
Z = specific side chain, which is in R form = none in T2 form = substituted GNAcyl in Ti form = (Galf)n, (Ribf)m in S(6,7) form = (Glc,, Man4, GNAc,). Abe in S(4,12) form = (-Man-Rha-Gal-)n Abe in SR(4,12) form = Man Rha-Gal FIG. 1. Schematic structure of the lipopolysaccharide of various Salmonella forms (1, 2, 7). Abbreviations: Glc, D-glucose; Gal, D-galactose; GNAc, N-acetylglucosamine; GNAcyl, N-acylglucosamine; Hep, heptose; KDO, ketodeoxyoctonate; Rib, D-ribose; Man, D-mannose; Abe, abequose; Rha, L-rhamnose. All sugars are pyranoses except those with an "f' suffix, which are furanoses.
(
FIG. 2. Simplified map of the Salmonella chroshowing the position of the genes and the characteristics of the donor (Hfr and F') strains used (14, 15).
inoculated previously with the appropriate bacteria in a soft-agar layer. Serological tests. Overnight bacterial growth from nutrient agar plates was used for agglutination. The tests were done on glass slides using 4% NaCl or antisera diluted appropriately in 0.2% NaCl containing 0.5% phenol as preservative. The antisera were prepared in rabbits as described by Kauffmann (5); the immunogen for anti-T2 serum was a heated (2 h at 100 C) suspension of SH 144. Crosses. Conjugation was the only method of genetic analysis used (15). Equal amounts of donor (Hfr or F') and recipient broth culture were mixed, and the mixture was incubated for 2 h without shaking. Aliquots (0.1 ml) of the mixture were then plated onto appropriate selective media. The recombinants appearing after 48 h of incubation were restreaked on nutrient agar plates, and single colonies from these were used for further tests. Culture media. Davis minimal medium (15) was used as selective medium with added agar (1.2%), glucose (0.2%), required amino acids (20 ,ug/ml), and/or streptomycin (1 mg/ml).
mosome
The second T2 line, SF 1187, was obtained from 0. Luderitz, Max-Planck-Institut fur Immunbiologie, Freiburg/Breisgau, Germany. Both strains were identical in agglutination tests and in their sensitivity to a range of S- or R-specific phages (see below). Mutants were selected from these strains after diethylsulfate treatment (9). Known Hfr and F' strains of Salmonella species were used as genetic donors (15). Phages. The rough-specific phage stocks were obtained from B. A. D. Stocker and propagated here; their properties have been described (24). When the sensitivity of a bacterial strain to phages was determined, phage lysates were applied as drops of about 108 plaque-forming units on a nutrient agar plate
RESULTS Properties of T2 strains. (i) Stability. When an overnight broth culture of a T2 strain was plated on nutrient agar, several of the developing colonies were morphologically rough, the rest appearing smooth. The agglutination reactions of these colony types confirmed them to be R and T2, respectively (Table 2). The R form was stable on subculture; the T2 form continued to segregate R colonies. The frequency of R forms in the overnight broth culture of T2 forms was approximately 5% and thus higher than in cultures of Ti strains. The formation of the R forms seemed to be a mutation from T2 positive to T2 negative. The same loss of the T2 antigen was later noticed in T2,S and T2,SR forms;
VOL. 13, 1976
1649
T2 LPS ANTIGEN OF SALMONELLA
TABLE 1. Salmonella strains used Strain no.
0
Species
C, C, C,
SF 1187 SH 144 SH 713 SH 1308 SH 1844 SH 1914 SH 2033 SH 3416 SH 5121
S. bareilly S. bareilly S. montevideo S. paratyphi B S. bareilly S. bareilly S. bareilly S. abony S. abony
SH 5152
S. abony hybrid B
B
C,
C, C, B B
Marker genesb
Mating type
Serotype
group'
Reference or parent strain
(4) (4) (8)
FFHfrH14 aro-852 leu-1357 met-1183 ilv-1123 FS59 Fhis-6143 Fhis-6143 gal-928 his-6143 FT1,S(1,4,5,12) HfrH1 met-1151 aro-851 str-501 HfrH2 met-1151 aro-851 S(1,4,5,12) his-6163 str-501 HfrH2 met-1151 aro-851 str-501 S(6,7) T2 HT2 S(6,7) Ti T2 R T2
(16) SF 1187 SH 1844 SH 1844 (15) SW 1403 from SH 713 x SH 5121
HfrH2 met-1151 aro-851 str-501 (15) B SW 1403 S. abony S(1,4,5,12) a The characteristic 0 antigens for group B are (1),4,(5),12; those for group C, are 6,7. b Standard gene symbols used (14). TABLE 2. Serological reactions and phage sensitivity of several T2+ and T2- forms of Salmonella bareilly compared with certain standard strains Sensitivity to phagesO Slide agglutination in:a Strain
4% AntiNaCi R
S. bareilly T2 R
Tic
~
++ -
S. typhimu-
T2
+
++
++ +
++
T2,S(6,7)r
S(6,7)r T2,SR(4,12) SR(4,12Y
Anti-
Anti- AntiTi 4,12
_
++ ++
Anti-
P22 FO 6SR
6,7P2FO6R
Br60 B6
Ffm Ffn
B
P221 2
X174
r
21C1OX7
+
+
+or-
+or-
-
-
+
+
+
+
+
-
-
+
+
+
-
-
+
+
+ or -
+
-
-
+
++
-
++
-
+
-
-
-
-
-
-
++
-
+
--
-
-
-
-
_ _
_ -
+ +
_ _
_ _
_ _
_ _
_ _
_ _
_ _
_
+
+
+
+
+
+
-
+
-
-
-
-
-
-
±
+ +
++ _-
_
+ +
++
++
++
++
+
riUMd
R(rfbi)
Tl
SR(4,12) S.
montevideo
+
-
++
-
-
-
+
+
+
+
+
+
-
+
±
+
-
-
+
-
-
+
-
-
-
-
+
-
-
++
++
++
+
+
_
+
+
+
+
+
-
-
ND ND ND
R (rfl) Tl
-
+
S(6,7)
-
-
++
-
-
|
-
-
-
+
+
+
+
+
-
-
++
-
+
-
-
-
-
-
-
a Antisera appropriately diluted in 0.2% NaCl. Symbols: + +, Strong agglutination with large clumps; +, strong agglutination, small clumps; ±, weak agglutination; -, no agglutination. b Phages applied as drops of 106 to 107 phages on nutrient agar plates previously spread with 0.1 ml of an overnight broth culture of the bacteria; incubated at 37 C. ND, Not done. Strains obtained as recombinants in crosses reported in this paper. d Several strains of each type described earlier (9, 16, 17). c
these mutated to S and SR, respectively (see below). (ii) Colony morphology. The T2-positive forms, either T2, T2,S, or T2,SR, were more opalescent than their Ti-negative mutants. This difference was more pronounced after longer incubation and at low temperature (20 C). The colony morphology and its dependence on temperature was reminiscent of the behavior of mucoid mutants. To exclude the possibility that the T2 forms were misinterpreted mucoid mutants, we isolated a set of true mucoid mutants and compared them with T2; they proved different both in their cultural
properties and the chemistry of the cell wall (M. Sarvas, M. Malinen, M. Nurminen, and P. H. Makel, submitted for publication). (iii) Agglutination reactions. Suspensions of the T2 forn were stable in 4% NaCl and agglutinated specifically, with formation of large clumps in anti-T2 serum (Table 2). In addition, the T2 form agglutinated to some extent in anti-R sera. For practical purposes the saline reaction was sufficient to differentiate T2 colonies from R colonies, which clumped strongly in saline and in anti-R, anti-T2, and anti-Ti sera and also to some extent in various (unabsorbed) anti-O sera. The strong reaction between R
1650
VALTONEN, SARVAS, AND MAKELA
colonies and anti-T2 sera may indicate the presence of anti-R antibodies in these sera, caused by the R mutants unavoidably present in the T2 batch used for immunization. Alternatively, this reaction may represent a cross-reaction between the R and T2 structures, but this could not be shown conclusively. (The same situation holds for the relation of R and Ti.) The T2,S(6,7) form found among recombinants (see below) agglutinated strongly and specifically in both anti-T2 and anti-0(6,7) sera, but not in saline, whereas its S(6,7) mutants agglutinated in anti-0(6,7) sera only. The T2,SR(4,12) form agglutinated strongly in anti0(4,12) sera, weakly in anti-R sera, and to a very slight degree in saline, whereas its SR(4,12) mutants agglutinated in anti-0(4,12) but not in anti-T2 sera and gave a slightly stronger reaction in anti-R sera and saline. A chemical examination of the LPS extracted from the various serologically classified forms confirmed the classification (2) into the various T2-positive and T2-negative forms. (iv) Phage sensitivity. When tested with a set of S- or R-specific phages (Table 2), the T2 form showed a sensitivity pattern closely resembling that known for R mutants with a complete LPS core (24). The R-specific phage P221 did not give plaques on the T2 strain, but since this phage did not grow on R mutants of the T2 strain either, it is assumed that this insensitivity was due to the fact that the T2 strains were derivatives of S. bareilly (O group Cl) rather than S. typhimurium (O group B), in which the phage pattems were originally determined (24). A similar nonsensitivity to some Rspecific phages has been noticed in R mutants of Salmonella groups Cl and L (9). In addition, the T2 form was completely resistant to phage Br2 and relatively resistant to the R-specific phage Ffm, whereas its R mutants were sensitive to both of them. It seems possible that the T2-specific structures prevent the access of these phages to their receptor sites in the LPS core while allowing some other Rspecific phages to attach. The phage sensitivity of the T2,S(6,7) and T2,SR(4,12) forms was similar to that of their T2-negative mutants. The SR(4,12) form was sensitive to phage FO only; although similar forms of Salmonella typhimurium are sensitive to both FO and P221, this S. bareilly derivative was not expected to be P221 sensitive (see above). Production of T2 derivatives with 0 specificity. Two main alternatives could account for the lack of 0 specificity in the T2 form. The first assumes that the genetic determinants of the T2 structure, called here rfu, are allelic to the
INFECT. IMMUN.
rtb cluster and then bacteria make either the rfu-directed T2 structure of the rtb-directed 0 side chain. The second assumes that rfu and rtb genes are independent of each other and the lack of 0 specificity in the T2 form is due to mutations of the rtb genes. This latter is the case in Ti forms in which rft genes determine Ti specificity (16). To decide between these alternatives, we replaced the rtb region of the T2 form with a functional rib from another, S strain; if recombinants with both T2 and 0-specific structures are found, the first alternative is excluded. This genetic exchange can be done by crossing a his- mutant of the T2 strain with a smooth donor strain and selecting recombinants that had received the donor his+ allele. Since the rib cluster is close to his, many his+ recombinants also receive the donor rib (8). When the S(1,4,5,12) HfrH2 donor SW 1403 (S. abony, 0 group B; see Fig. 2) was in this way crossed with the T2 his- recipient SH 1844 (crosses A and B, Table 3), about 40% of the his+ recombinants showed the donor 0 specificity 4,12, as expected (0 factors 1 and 5, also present in the donor strain but not determined by rfb genes, were not tested). These recombinants also agglutinated in anti-R sera and to a slight degree in saline in a way characteristic of SR forms. This is the form expected in crosses of group B donors with group C, recipients (8) in which the recombinants receive the rib genes of the donor but not the remote rfc gene(s) that would be required for the polymerization of the donor-type 0-specific units. Most of these SR(4,12) recombinants were also agglutinated in the anti-T2 serum, indicating that they had retained the rfu genes determining T2 specificity. In cross B, three out of the 18 recombinants with 0-4,12 appeared to have lost the T2 specificity. It was not possible to find outAvhether they had lost their rfu+ genes in the recombination process or were the result of a mutation from T2 positive to T2 negative (in the recipient or the recombinant). In either case, the results show that rfu is not allelic or even close to the rib. The lack of 0 antigen in the T2 form must hence be due to an rtb defect(s). For comparison, a T2-negative mutant of the T2 recipient parent in crosses A and B was used as recipient in a similar cross with the same donor (cross C, Table 3). Now 30% of the 35 his+ recombinants had the donor 0 specificity; although most of these recombinants were SR(4,12), one was S(4,12), indicating that it had received both the rtb and rfc genes from the donor. No T2-positive forms were obtained. Because S. bareilly is of group C,, characterized by the 0 specificity 6,7, we also wanted to
T2 LPS ANTIGEN OF SALMONELLA
VOL. 13, 1976
1651
TABLE 3. Analysis ofthe his+ recombinants in crosses between a his+ met- S(4,12) Salmonella abony donor SW 1403 and his- met' S. bareilly recipients, either T2 or R Cross
Serotype
Donor S. abony SW 1403 Recipient S. bareilly SH 1844 his+ recombinants: 52 in cross A l 42 in cross B
S(4,12) T2 T2 T2,SR(4,12) SR(4,12)
Genes involved in the crosses"
Percent ofeach recombinant class cross A cross B
hibs +
rfb +(4,12)
D D D
r D D
d
60 40 0
57 36 7
rfu' + r r D or r'
rfc +
metP
_C
+ r r r
ND r r
+ _ _ _ R r ND r D 71" R r r D D 26" SR(4,12) r D D D 39 S(4,12) a Abbreviations: D, Donor allele; r, recipient allele for each gene locus or cluster tested; ND, not determined. b Markers used for the selection of recombinant. rfu stands here for any gene required for the production of the T2 antigen. d Inferred from the outcome of the cross. " rfc+ together with rfb+ (4,12) leads to an S(4,12) serotype, whereas rfc- with rfb+ (4,12) leads to SR(4,12). The rfc character cannot be assessed with rtb- or rfb+ (6,7), but was deduced from the behavior of the recombinants produced. f rfu- can be a donor character or a recipient rfu+ that had mutated to rfu-. D Results for cross C.
Recipient S. bareilly SH 1914 his+ recombinants 35 in cross C
"
check whether the T2 strain could make the 06,7 antigen if given complete rib genes of this 0 type. The yield of recombinants with an 0-6,7 HfrH14 donor, SH 713 of group C,, was very low, however. An 0-6,7 derivative SH 5152 of the HfrH2 strain used in crosses A and B was a better donor, probably because it transfers his and rib efficiently close to its origin. Among 38 his+ recombinants isolated (SH 5152 x SH 1844), 22 were T2, nine were T2,S(6,7), four were S(6,7), and three were R. It seems probable that the S and R recombinants were derived from T2-negative mutants of the recipient. The production of T2,S(6,7) recombinants showed, however, that the T2 strain was capable of synthesizing the 0-6,7-type side chain when it was provided with functional rib genes of this type. Attempts to investigate the relation of Ti and T2 determinants and to localize the rfu gene(s). We crossed a Ti-positive S. paratyphi B strain SH 1308 (rft+ rfb+ ilv- met-) containing the episome FS59 as donor to the T2-positive strain SH 2033 (rfu+ gal- his- rib-) as recipient, and selected the gal+ ilv+ met+ recombinants. The frequency of recombinants was low, approximately 3 x 10-7. Of 90 recombinants obtained in two separate crosses, 88 agglutinated like T2, and two agglutinated like Ti. No recombinants with both T2 and Ti specificity were found. In further crosses the donor was SH 3416, a Ti-positive derivative of S. abony HfrHl; the recipient was the same SH 2033. Of 158 gal+ recombinants, two were Ti, nine were R, and 147 were T2. No strains with
both Ti and T2 were seen. The Ti-positive recombinants probably had received the donor rft+ genes; their frequency (4/248) was very much lower than expected on the basis of the previously observed approximately 50%o linkage between rft and gal. We believe that this poor linkage is due to low homology between the parents in these crosses
(15). The absence of T1,T2 recombinants might suggest that Ti and T2 are determined by allelic genes. It is also possible that either Ti or T2 is epistatic to the other. The small number of Ti-positive recombinants reduces the value of these crosses; it is possible that recombinants were preferentially formed from T-negative mutants of either donor or recipient (or both) so that no recombinants would be expected to have rft+ and rfu+ The poor recombination frequency, low linkage, and frequency of T2-negative mutants in the T2 strains proved prohibitive to attempts at localizing the rfu genes. More conclusive results would be expected from crosses using T2positive donors, but all our attempts at producing such were unsuccessful, e.g., all recombinants from the above-mentioned crosses between F' donors and T2 recipients were F-. DISCUSSION The Ti form has been previously shown to be genetically rft+ and ri- and to mutate at high frequency to the Tl-negative form rft- rib-. (We have chosen to use rft+ and rfu + as the
1652
VALTONEN, SARVAS, AND MAKELA
symbols for the uncommon form of postulated genes determining the uncommon antigens Ti and T2 and the corresponding symbols with superscript "minus" for the common form, not determining any such antigen. In this way "plus" is connected with a functional form and "minus" is connected with a nonfunctional form; note that this is opposite of the usage specified in the standard convention of bacterial genetics.) The rib- of the Ti form could be replaced by a functional rfb gene cluster to produce T1,S or T1,SR forms containing both Ti and 0 side chains in their LPS and showing both Ti and 0 serological specificity (16, 17). Furthermore, both the Ti and 0 side chains have been shown to be attached to the LPS core in the same position, carbon 4 of the terminal glucose, which carries N-acetylglucosamine at carbon 2 (1). The T2 form seems to be analogous to Ti in all these respects. The T2 form isolated from a natural source is genetically rfu+ rfb- and mutates to R (rfu-). By genetic manipulation, T2,S and T2,SR forms (rfu+ rfb+) can be derived from it; these can in their turn mutate to rfu- at the same high rate as T2 mutates to R. The T2 chemical determinant (a substituted N-acylglucosamine) is linked to carbon 4 of the glucose II of the LPS core in the same way as are 0 and Ti side chains (2) (Fig. 1). In a T2,SR form, the amount of 0-specific material was reduced compared with its T2-negative (that is, SR) mutant, suggesting competition between the T2 and 0 determinants (2). This outcome is expected since they share the same attachment site. The Ti determinant is a long polysaccharide chain, whereas T2 specificity is based on a substituted monosaccharide only. The long Ti side chains are apparently relatively few in number; consequently they do not "cover up" the rest of the LPS very efficiently, and the Ti form is nearly as sensitive to the R-specific phages as is its Ti-negative mutant. The T2 determinant occupies a larger fraction of the core stubs. In the study of Bruneteau et al. (2), more than 50% of core stubs in T2 form LPS had the T2 determinant N-acylglucosamine; the proportion could not be determined exactly because of the variable occurrence of R mutants in the culture batches from which the LPS was extracted. In the same study the T2,SR form showed a ratio of 0 units to core to be 1:2 (calculated from a 1:4 ratio of rhamnose to glucose) in a batch in which we determined the frequency of T2-negative SR mutants to be exceptionally low, approximatively 5%. As the 0 unit-to-core ratio in the corresponding SR form was 1:1, this would indicate that 50%o of the core
INFECT. IMMUN.
stubs were substituted by the T2 unit. In this paper (Table 2) we saw that the presence of T2 determinants reduces the sensitivity of the T2 form to several R-specific phages compared with its R mutants. An analogous situation has been seen in SR forms in which every core stub is covered by a trisaccharide 0 unit and which are resistant to most of the R-phages (24). ACKNOWLEDGMENTS We thank Seijasisko Suurmnkki for skillful technical assistance. This research was supported by Public Health Service grant GM-12046 from the National Institute of General Medical Sciences and by grants from the Finnish Medical Research council and Sigrid Juselius Foundation. LITERATURE CITED 1. Berst, M., C. G. Hellerqvist, B. Lindberg, 0. Luderitz, S. Svensson, and 0. Westphal. 1969. Structural investigation on TI lipopolysaccharides. Eur. J. Biochem. 11:353-359. 2. Bruneteau, M., W. A. Volk, P. P. Singh, and 0. Luderitz. 1974. Structural investigations on theSalmonella T2 lipopolysaccharide. Eur. J. Biochem. 43:501-508. 3. Kauffmann, F. 1956. A new antigen of Salmonella paratyphi B and Salmonella typhimurium. Acta Pathol. Microbiol. Scand. 39:299-304. 4. Kauffmann, F. 1957. On the T antigen of Salmonella bareilly. Acta Pathol. Microbiol. Scand. 40:343-344. 5. Kauffmann, F. 1966. The bacteriology of enterobacteriaceae. Munksgaard, Copenhagen. 6. Lindberg, A. A., M. Sa.rvas, and P. H. Makela. 1970. Bacteriophage attachment to the somatic antigen of Salmonella: effect of 0-specific structures in leaky R mutants and S, Ti hybrids. Infect. Immun. 1:88-97. 7. Ltideritz, O., 0. Westphal, A. M. Staub, and H. Nikaido. 1971. Isolation and chemical and immunological characterization of bacterial lipopolysaccharides, p. 145-234 In G. Weinbaum, S. Kadis, and A. J. Ajl (ed.), Microbial toxins, vol. IV. Academic Press Inc., New York. 8. Mrikela, P. H. 1966. Genetic determination of the 0 antigens of Salmonella groups B (4,5,12) and C, (6,7). J. Bacteriol. 91:1115-1125. 9. Mikela, P. H., M. Jahkola, and 0. Ltideritz. 1970. A new gene cluster rfe concerned with the biosynthesis of Salmonella lipopolysaccharide. J. Gen. Microbiol. 60:91-106. 10. Mikela, P. H., and B. A. D. Stocker. 1969. Genetics of polysaccharide biosynthesis. Annu. Rev. Genet. 3:291-322. 11. Nikaido, H., and M. Sarvas. 1971. Biosynthesis of the Ti antigen in Salmonella: Biosynthesis on a cell-free system. J. Bacteriol. 105:1073-1082. 12. Roantree, R. J. 1971. The relationship of lipopolysaccharide structure to bacterial virulence, p. 1-38 In G. Weinbaum, S. Kadis, and A. J. Ajl (ed.), Microbial toxins, vol. IV. Academic Press Inc., New York. 13. Ruschman, E., and 0. Luderitz. 1971. The virulence for mice of Salmonella typhimurium Ti mutants. Zentralbl. Bakteriol. Parasitenkd. Infektronskr. Hyg. I Abt. Orig. 216:185-192. 14. Sanderson, K. E. 1972. The linkage map of Salmonella typhimurium. Bacteriol. Rev. 36:558-586. 15. Sanderson, K. E., H. Ross, L. Ziegler, and P. H. Mikelii. 1972. F+, Hfr, and F' strains of Salmonella
VOL. 13, 1976
typhimurium and Salmonella abony. Bacteriol. Rev. 36:608-637. 16. Sarvas, M. 1967. Inheritance of Salmonella Ti antigen. Ann. Med. Exp. Fenn. 45:447-471. 17. Sarvas, M., and P. H. Makelai. 1965. The production, by recombination, of Salmonella forms with both Ti and 0-specificities. Acta Pathol. Microbiol. Scand. 65:654-656. 18. Sarvas, M. and H. Nikaido. 1971. Biosynthesis of Ti antigen in Salmonella: Origin of D-galactofuranose and D-ribofuranose residues. J. Bacteriol. 105:1063-
T2 LPS ANTIGEN OF SALMONELLA
aspects of biosynthesis and structure of Salmonella lipopolysaccharide, p. 369-438. In G. Weinbaum, S.
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