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Journal of General Microbiology (1g77), roo, 355-361
Printed in Great Britain
355
Genetic Determinants of the Synthesis of the Polysaccharide
Capsular Antigen K27(A) of Escherichia coli
By G. SCHMIDT, BARBARA J A N N A N D K. J A N N
Max-Planck-lnstitutfur Immunbiologie, D 7800, Freiburg, Germany
I D A 0 R S K O V A N D F. 0 R S K O V
Statens Seruminstitut, WHO Collaborative Centrefor Reference and Research
on Escherichia, DK 2300, Copenhagen, Denmark
(Received 22 November 1976; revised 8 February 1977)
SUMMARY
Most of the his+ hybrids from crosses between the Escherichia coli donor
Hfr45(08 :K27) and different E. coli Og recipients expressed the donor 0 8 antigen
specificity and produced the capsular antigen K27. Therefore these hybrids must
have inherited the his-linked donor rfb region determining the synthesis of 08specific polysaccharides as well as his-linked genes involved in K27 antigen
synthesis. In the living state these hybrids were inagglutinable in 0 8 antiserum like
the donor cells. However, when E. coli K I and
~ 0 8 : Q 2 - were used as recipients
most of the his+ hybrids were agglutinable in 0 8 and K27 antisera. The amounts
of K27 antigen present in these hybrids, designated as K27i (intermediate) forms,
were sufficient to evoke the production of K27 antibodies in rabbits, but insufficient
to inhibit 0-agglutination of the respective cells. The additional transfer of the trp
region of E. coli 0 8 :K27 into such K27i forms frequently resulted in 0-inagglutinable K27f hybrids. This is attributed to the introduction of trp-linked genes which
apparently play a role in the synthesis of K27 capsular antigen. Thus it is concluded
that at least two gene loci, one close to his and the other close to trp, are required
for the synthesis of the complete capsular antigen K27.
INTRODUCTION
Many Escherichia coli serotypes produce capsular antigens (K antigens) which usually
consist of acidic polysaccharides (Jann & Westphal, 1975). A strong indication of the
presence of K antigens is the inagglutinability of living bacteria in homologous 0 antiserum
(Kauffman, I 966). Differences in the temperatures required to overcome this 0-inagglutinability and in the thermoresistance of K antibody-binding and agglutinating capacities have
been decisive for the division of K antigens into three types, the A, B and L antigens
{Kauffmann & Vahlne, 1945; Kauffmann, 1966). The A antigens are relatively thermostable
capsules which, in contrast to B and L antigens, still inhibit agglutination of the bacteria in
homologous 0 antiserum after heating at IOO "C for 2-5 h. Strains with K(A) antigens have
to be autoclaved to make them agglutinable in 0 antiserum.
Although in recent years there has been considerable progress in understanding the nature
and role of K antigens (0rskov et al., 1971; Kaijser, 1973; McCabe et al., 1975; Nicholson &
Glynn, 1g75), knowledge of their genetics is scanty. Recently, a gene locus participating in
the synthesis of the K(L) antigens KI, K4, KIOand K54 has been found closely linked to
serA and was termed kpsA (0rskov & Nyman, 1974; 0rskov, Sharma & 0rskov, 1976).
Another gene apparently needed for the expression of these K(L) antigens has not yet been
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356
G. S C H M I D T A N D OTHERS
Table
Strain
I.
Characteristics of E. coli strains
Derived from
Serotype
Relevant genotypet
Donor
Hfr4o(cw)*
~56b
0 8 :K27
Hfr45(ccw)
~56b
0 8 :K27
XYl
KL25(CW)
K12
Rough
thi
Recipient
F7 1
~56b
0 8 :K27
his trp ara lac str
F464
~56b
0 8 :K27his met pro ara mtl str
F713
Bil61142
0 9 :K29his trp str
~720
SU397314I
09:K3 I his str
F730
A295b
0 8 :K42his trp ara str
2402
E69
O9:K30his str
2578
K12
Rough
his trp str
* cwlccw, Clockwise/counterclockwisedirection of transfer.
t Genes for his, histidine; met, methionine; pro, proline; thi, thiamin biosynthesis: ara, arabinose; lac,
lactose; mtl, mannitol; xyl, xylose utilization: str, streptomycin resistance.
mapped. Genes participating in the synthesis of the K(A) antigen of E. coli Og :K26 (Orskov
& Orskov, 1962) are located close to the his-linked rfb region which is involved in the
synthesis of 0 antigens (Stocker & Makela, 1971). This paper describes experiments demonstrating that, in addition to this his-linked gene, a second trp-linked gene is needed for the
synthesis of the complete K27(A) antigen of E. coli 0 8 :K27.
METHODS
Bacterial strains. These are listed in Table I. The donor strains Hfr4o and Hfr45 have
their points of origin for the chromosome transfer near xyl and his, respectively (Schmidt,
Jann & Jann, 1970). The E. coli K 1 2 donor ~ ~ transfers
2 5 its chromosome with ilv as a
leading marker (Low, 1968).
Media. D1,,-agar (Schlecht & Westphal, 1967) and Merck Standard I broth were used
as complete media. Recombinants were selected on minimal agar (Lederberg, 1950) containing streptomycin (100,ug ml-l) to inhibit the growth of the donors. When required,
amino acids were added to a final concentration of 20 ,ug ml-l.
Mating experiments. These were performed as described previously (Schmidt et al., I 970).
Recombinant clones were first transferred on to the same medium used for primary selection
and then streaked on to complete agar from which single colonies were isolated.
Serologicalmethods. The purified recombinants were scored for their antigenic properties
by both slide and tube agglutination tests (Kauffmann, 1966). Absorption of antisera with
whole bacteria was performed as described previously (Mayer & Schmidt, 1973). Some
recombinants were also examined by immunoelectrophoresis.
Antisera. Rabbit 0 antisera were prepared as described previously (Mayer & Schmidt,
1973) using boiled suspensions of non-capsulated E. coli strains (08 :K27- and Og :K29-)
as antigens. K27 antiserum was obtained by immunization with a formalin-treated suspension of E. coli ~ 7 8 2which
,
is a rough form (08-) encapsulated with K27 antigen (Whang
et al., 1972).
Imrnunoelectrophoresis. This was done as described by Scheidegger (I 955) using extracts
prepared as described by Orskov et al. (1971).
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Genetic determinants of K antigens
357
Table 2. Serological analysis of his+ recombinants selected from crosses between the donor
Hfr45(08 :K27) and various acapsular recipients
Recipient
Serotype of
recipient
No. of
recombinants
analysed
Serological types identified*
\
A
O8:K-
0g:K-
K27
Rough
K27i
I12
8
O8:K270
104
0
0
F464
F7I 3
Og :K2g7
86
0
0
I00
7
~720
Og:K318
87
0
0
I00
5
2402
0 9 :K3o9
91
0
0
I00
0
F730
0 8 :K42257
I0
0
7
0
240
Rough (K12)
0
3
8
109
2578
I22
2
* O8:K-, agglutinable in 0 8 antiserum, non-reactive in K27 antiserum. O9:K-, agglutinable in Og
antiserum, non-reactive in K27 antiserum. K27, agglutinable in K27 antiserum, non-reactive in 0 antisera.
Rough, unspecific agglutination in 3.5 % saline. K27i, agglutinable in 0 8 and K27 antisera.
Table 3. Inheritance of trp and serologicalpatterns of 257 his+ hybridsfrom the cross
between the donor Hfr45(08 ;K27) and F730(08 :K42-)
his+ hybrids
with
K forms*
No.
r
K27-
A
K27i
\
K27+
248
9
238
I
Recipient trp
9
I
2
6
Donor trp+
Total
257
I0
240
7
* K27-, agglutinable in 0 8 antiserum, non-reactivein K27 antiserum. K27i, agglutinable in K27 and 0 8
antisera. K27+, agglutinable in K27 antiserum, non-reactivein 0 8 antiserum.
RESULTS
Genetic transfer of the K27 antigen
Earlier studies demonstrated a close linkage between the his operon and genetic determinants of the K26(A) antigen (Orskov & 0rskov, 1962) in genetic crosses. Recent experiments have suggested that a similar situation exists with regard to the K27(A) antigen
(Olson, Schmidt & Jann, 1969). To confirm this we crossed the E. coli donor Hfr45(08 :K27)
with several acapsular (K27-, K29-, K ~ o - ,K31- or K42-) his recipients of 0 8 or 0 9
serotypes and the rough K 1 2 strain and selected recombinants which had received the his+
allele of the donor. The his+ hybrids were divided on the basis of slide agglutination tests
into several serological types (Table 2).
Most of the his+ hybrids derived from crosses with the K- mutant ~464(08:K27-)or
the three Og :K- strains as recipients were inagglutinable in the 0 antisera, but were strongly
agglutinated in K27 antiserum, Thus these hybrids behave like K27+ forms and consequently must have inherited the genetic information for K27 antigen synthesis together with
the donor his+ allele. In crosses with Og recipients the K27+ hybrids synthesized the 0 8
antigen instead of the recipient’s Og antigen, as detected by the sensitivity of the hybrids
to the 0 8 antigen-specific phage Q8 (Jann et al., 1971). Accordingly, the his-linked donor rfb
region responsible for 08-specific polysaccharide synthesis had been introduced together
with the ‘K27’ genes into the hybrids.
Using strains ~ 7 3 0(K- of 0 8 : Q 2 ) and 2578 (KI2) as recipients an additional hybrid
class was found which comprised the majority of the his+ hybrids. These hybrids were
agglutinated in 0 8 as well as in K27 antisera (Table 2). The reactivity in K27 antiserum
indicates the presence of K27 antigen. The 0-agglutinability, however, suggests that the
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G. S C H M I D T A N D OTHERS
Table 4. Serological analysis of trp recombinants selected from crosses bet ween
the donor Hfr4o and K27i recipients
f
K forms*
K27i
recipient
Dzrived
from
No. of trpf
recombinants
analysed
K27
K27i
K-
F733
2592
F730
2578
I27
62
91
52
34
8
2
2
r
\
A
* K27, agglutinable in K27 antiserum, non-reactive in 0 8 antiserum. K27i, agglutinable in K27 and 0 8
antissa. K-, agglutinable in 0 8 antiserum, non-reactive in K27 antiserum.
Table 5. Reciprocal agglutination titres of K27, K27i and K27- bacteria in
various antisera
~777(08:K27i) antiserum
Bacteria for
agglutination
&
I
Unabsorbed
~ 7 8 2 6 2 7 antiserum
)
\
Absorbed with
E. coli 0 8 :K27-
Unabsorbed
Live or formalin-treated cultures
80
80
320
F7IW27)
F492627-1
25120
< 20
< 20
2592cK27i)
2 2560
(I 280)*
( 22560)
F7336 2 7 9
2 2560
(1280)
( 22560)
F955-79
2 2560
( 22560)
(B 2560)
Heat-killed cultures?
I 60
80
640
F7I W27)
20
< 20
25120
F492627-)
< 20
< 20
25120
25 92(K270
< 20
< 20
25120
F733cK2 70
< 20
< 20
25120
F955cK27i)
* Titres in parentheses indicate finely grained agglutinates.
t Bacterial susp2nsions were heated for I h at 100 "Cand washed once with saline.
hybrids are intermediate between normal K27+ and K27- forms. Therefore these hybrids
were designated as K27 intermediate forms, abbreviated as K27i (Table 2). Only 7 out of
257 hybrids from a cross with the recipient ~ 7 3 0were inagglutinable in 0 8 antiserum like
K27+forms. A detailed analysis of these hybrids (Table 3) showed that nine had also received
the non-selected trpf from the donor; among those were six of the seven K27f hybrids. A
similar result was obtained with the E. coli K I 2 recipient 2578 (Table 2). The majority of
the his+ hybrids were K27i forms agglutinable in 0 8 antiserum, indicating that they had
also received the donor rfb region. Two of the three K27+ hybrids found among the his+
hybrids were trpf. All the other hybrids had retained the Trp- phenotype of the recipient.
These results suggested that synthesis of the complete K27 antigen requires gene(s) in
the trp region in addition to the his-linked genes. This was confirmed when the trp defect in
K27i hybrids derived from crosses with E. coli ~ 7 3 0and 2578 was cured in crosses with the
E. coli donor Hfr4o : 70 to 80 % of the trp+ hybrids behaved like normal K27+forms (Table 4).
Transfer of the trp1regionfrom E. coli KI 2 to E. coli 0 8 :K27
If the E. coli KI 2 trp region is introduced into E. coli 0 8 :K27 recipients, one would expect
some of these hybrids to be K27i forms as a result of the substitution of trp-linked 'K27'
genes by an allelic KI 2 region lacking these determinants. Therefore we crossed the E. coli
K I donor
~
~ ~ with
2 the
5 recipient F ~ (08
I :K27, his, trp, ara, lac) and selected trp+ hybrids.
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Genetic determinants of K antigens
359
All the 128 trpf recombinants had retained the His- phenotype of the recipient and about
10% of them had incorporated donor genes for arabinose and lactose utilization. Of the
128 trp+ recombinants, 94 (73%) behaved like K27i forms, while 32 were K27f like the
recipient, and two were K27- (still 08.).
Serologicalproperties of Ki hybrids
To further characterize the K27i hybrids we performed tube agglutination tests with
heat-killed (roo "C,I h) and formalin-treated cultures. The heat-killed bacterial antigen
suspensions were washed once with saline. We used ~782(K27)antiserum and an OK antiserum produced with a formalin-treated suspension of a K27i hybrid (~777,0 8 :K27i).
From the latter serum a pure K antiserum was obtained by absorption with a heated culture
of E. coli 0 8 :K27-. The reciprocal agglutination titres of some K27i and K27 hybrids from
different crosses and of normal K27 forms in various sera are given in Table 5. The titres
of I :80 with formalin-treated and of I :80 to I : 160 with heated K27 bacterial suspensions
in F777 antiserum showed that the K27i form can evoke the production of K27 antibodies.
It was striking that in K27 antiserum the formalin-treated K27i hybrids formed very finegrained agglutinates compared with the heavy conglomerates of K27 cells. Similar observations were made in slide agglutination tests. The K27i forms gave much higher K titres than
K27 cells. The titres obtained with formalin-treated K27i cells in F777(0K) antiserum were
certainly due to mixed K and 0 agglutination.
Another difference between K27i forms and K27 forms became evident when heated,
washed cell suspensions were used for agglutination tests. In contrast to K27 cells the heated
K27i forms had lost their agglutinability in pure K27 serum. This may be due to a loss of
K antigen from the pretreated cells. Thus the reactivity of heated K27i hybrids in unabsorbed
F777(oK) antiserum must be an 0 antigen agglutination.
It is known that the K27 antigen retains its antibody-binding capacity after heat treatment
(Kauffmann, 1966). To see whether this was also true for K27i hybrids, we absorbed K27
antiserum with heated and living cells of K27i hybrids and tested for any remaining K27
antibody in the absorbed sera. In no case did we observe agglutination with formalin-treated
cultures of K27f cells. This indicated that the K antibody-binding capacity of K27i forms
was not abolished by heat treatment.
Two antisera prepared with formalin-treated suspensions of two K27i hybrids which had
received the his but not the trp marker from the donor were tested by immunoelectrophoresis.
In both antisera both 0 and K precipitation lines (0rskov et al., 1971) were formed with
extracts of E. coli 08:K27 and of two K27 recombinants to which the his and the trp loci
had been transferred. In contrast, extracts from the K27i cultures used for immunization
showed only an 0 antigen precipitation line and no K precipitation line.
DISCUSSION
The K27 antigen is an acidic polysaccharide, the repeating units of which consist of
glucose, galactose, fucose and glucuronic acid (Jann, Jann & Schneider, 1968). As shown
above, two gene loci appear to participate in the synthesis of the K27 antigen of E. coN
0 8 :K27, one closely linked to his, the other to trp. The serological results suggest that lack
of the trp-linked K27 gene(s) led to a reduced expression of the K27 antigen, as judged from
the 0-agglutinability of such hybrids and the fact that the Kq-specific precipitation line
was absent in their immune electrophoretic pattern. Because of the intermediate position
of such hybrids between K27+ and K27- forms, they were designated as intermediate
forms (K27i).
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360
G. S C H M I D T A N D OTHERS
In matings with K- recipients derived from E. coli Og :K3o and Og :K3 I it was sufficient
to introduce the his-linked genes to obtain hybrids with complete K27 antigen. This means
that these recipients must have the trp-linked genes which are required for the synthesis of
the complete K27 antigen. Like E. coli 0 8 : K27, the E. coli Og strains mentioned above have
glucuronic acid as a constituent of their K antigens (Jann & Westphal, 1975). Escherichia
coli 0 8 :K42, which lacks the trp-linked gene needed for complete K27 antigen synthesis,
contains galacturonic acid in its K antigen (Jann & Westphal, 1975). Thus it appears possible
that the trp-linked locus is somehow connected with glucuronic acid, the acidic constituent
of the K27 and other K antigens.
It is not likely that the product of the trp-linked 'K27' gene acts on the level of glucuronic
acid precursors because UDPglucuronic acid is a precursor of UDPgalacturonic acid, which
in turn is needed for K42 antigen synthesis. However, such a gene product could possibly
be a polymerase (or a subunit of such an enzyme) which recognizes glucuronic acid as a
reactive site. The trp-linked 'K27' gene would then correspond to the Salmonella rfc locus
which controls the polymerization of 0-specific repeating units in certain Salmonella strains
(Stocker & Makela, 1971).
If this were true, the K27-specificity of K27i cells would not be due to a polysaccharide,
but only to single oligosaccharide repeating units linked to an acceptor (possibly core-lipid
A). It is conceivable that the transfer of one repeating unit in K27i cells is less effective than
that of the wild-type polysaccharide in K27 cells. Thus the K27 antigen of K27i forms would
differ from that of the wild-type K27 strain quantitatively and qualitatively. This reduction
of the K27 antigen in size and amount might explain the 0-agglutinability of K27i cells and
the lack of the K27i precipitation line in immune electrophoresis.
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