O-antigen variation in Salmonella spp.: rfb gene clusters of three strains.

Abstract

The O antigens of Salmonella serogroups A, B, and D differ structurally in their side-chain sugar residue. These genes encoding O-antigen biosynthesis are clustered in the rfb operon. We report here the molecular cloning and analysis of the rfb operons of Salmonella paratyphi A (serogroup A) and S. typhi (serogroup D). The regions of DNA nonhomology between the rfb operons of these serogroup A, B, and D representatives are identified, and the evolutionary derivation of serogroup A from a serogroup D progenitor is discussed.

Full text

Page 1

JOURNAL OF BACTERIOLOGY, Jan. 1988, p. 103-107
0021-9193/88/010103-05$02.00/0
Copyright C 1988, American Society for Microbiology
Vol. 170, No. 1
O-Antigen Variation in Salmonella spp.: rfb Gene Clusters of
Three Strains
NARESH K. VERMA,* NEIL B. QUIGLEY, AND PETER R. REEVES
Department of Microbiology, the University ofSydney, Sydney, New South Wales 2006, Australia
Received 20 April 1987/Accepted 23 September 1987
The 0 antigens of Salmonella serogroups A, B, and D differ structurally in their side-chain sugar residue.
The genes encoding 0-antigen biosynthesis are clustered in therjboperon. We report here the molecular
cloning and analysis oftherjboperons ofSalmonellaparatyphiA (serogroup A) and S. typhi (serogroup D). The
regions of DNA nonhomology between therjb operons of these serogroup A, B, and D representatives are
identified, and the evolutionary derivation of serogroup A from a serogroup D progenitor is discussed.
Lipopolysaccharides are integral components of the outer
membranes of gram-negative bacteria and generally consist
of three structural regions: the lipid A moiety, which is
hydrophobic and is embedded in the outer membrane, an
oligosaccharide core, and a polysaccharide chain commonly
known as the 0 antigen. The 0-antigen chains ofSalmonella
typhi, S. paratyphi A, and S. typhimurium, representative of
Salmonella serogroups D, A, and B respectively, have
identical trisaccharide subunit backbones (mannosyl-rham-
nosyl-galactosyl). These chains can be distinguished, since
each has a different 3,6-dideoxyhexose attached to the
mannosyl residues as a side branch. The side branch is
tyvelose in S. typhi, paratose in S. paratyphi A, and abe-
quose in S. typhimurium (11, 13). Much of the antigenic
variation among Salmonella species is the result of genetic
variation in the rfbgene cluster, which maps at 42 min on the
chromosome of S. typhimurium LT2 (17). We previously
reported the cloning and restriction analysis oftherfbcluster
from S. typhimurium LT2 (1, 2). We report here the cloning
and analysis of the rfb loci from S. typhi and S. paratyphi A
strains and compare them with the S. typhimurium rfblocus.
We have used the traditional taxonomy for Salmonella
species, although as Brenner (3) points out, the statement in
the 1974 (8th) edition ofBergey's Manual that "Scientifically
none of the present methods of nomenclature of Salmonella
is satisfactory" is as true today as more than a decade ago.
It is clear that all Salmonella strains are in one species (3, 7),
and Ewing (7) has used a single species name, although the
International Subcommittee has still not made a recommen-
dation. It is important to note that the strains of S. typhimu-
rium, S. paratyphi A, and S. typhi used in this study are in
reality not only members of the same species, but also of the
same subspecies, and the homology and nonhomology we
describe for different regions of the rfb gene cluster must be
seen in this light.
MATERIALS AND METHODS
Bacteria and plasmids. Strains used are listed in Table 1. A
series of plasmids which carry DNA spanning the entire rfb
operon of S. typhimurium LT2 (2) were used in this study.
Enzymes and radiochemicals. All enzymes and radioche-
micals used in this study were obtained as described previ-
ously (2).
DNA techniques. The methods used for DNA preparation,
*Corresponding author.
agarose gel electrophoresis, radioactive labeling of DNA, in
situ DNA hybridization, autoradiography, ligation, and bac-
terial transformation were those described by Maniatis et al.
(14). Molecular size standards were used as described in the
companion paper (2).
Cosmid cloning. Sau3A partial digests of chromosomal
DNA from S. typhi Ty2la and S. paratyphi A IMVS1316
were prepared by the method of Maniatis et al. (14). The
cosmid used in this experiment was pHC79 (10). It was
digested with BamHI and ligated with the partial Sau3A
fragments in a final volume of 20 ,u by mixing 2 ,ug of insert
DNA with BamHI-digested and phosphatase-treated vector
DNA in a molar ratio of 1:3. A 3-pI portion of this ligation
mixture was packaged in vitro by the method of Stemnberg et
al. (19), with a commercially available packaging kit (Pro-
mega 13iotec, Madison, Wis.). DH1 was used as the host
strain for infection and was plated out on ampicillin agar
(containing 100 pug of ampicillin per ml). 2 x 104 recombi-
nants per ,ug of ligated S. typhi DNA and 103 recombinants
per pug of ligated S. paratyphi DNA were recovered.
Preparation of heteroduplex DNA and electron microscopy.
Heteroduplex formation and DNA spreading were per-
formed by the method ofDavis and Parkinson (6) with minor
modifications. Grids were prepared by the basic protein film
technique (12) and stained with uranyl acetate or rotary
shadowed with platinum-palladium.
RESULTS
A 2.25-kilobase (kb) KpnI fragment, which lies approxi-
mately in the middle of the S. typhimurium rib operon
(positions 9.95 to 12.20), was isolated from pPR300 (2) and
used as a radioactive probe to screen cosmid libraries for the
presence of homologous DNA. Eight clones in the S. typhi
Ty2la library and two clones
IMVS1316 library were identified with this probe. Cosmid
DNAs from these 10 clones were digested with EcoRI and
run on agarose gels; they were found to have a number qf
common bands, many ofwhich also occur in S. typhimurium
(2). These fragments were assumed to be homologous to
those of S. typhimurium, and on this basis partial mapsof the
cosmids were deduced (Fig. 1).
Hybridization of the same 2.25-kb radioactive probe with
blotted EcoRI digests of all cosmids and with chromosomal
DNA from S. typhi Ty2la and S. paratyphi A IMVS1316
identified a single fragment in each case which, with the
exception of pPR429, had the same mobility as the 11.02-kb
in the
S. paratyphi A
103

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104VERMA ET AL.
TABLE 1. Bacterial strains
Strain
Characteristics
Source or reference
E. coli K-12 DEll
S. typhimurium LT2 SL1654
supE44 gyrA96 recA endAI thi-J hsdRJ7 relAl
hsdL6 trpC2 nml Hlb fla-66 H2-enx rpsLl20 xy1404 ilv452 metE55
metA22 hsdSA29
9
I. Beacham
S. paratyphi A IMVS1316
S. typhi Ty2 Ty2la
S. Dixon
8
galE
EcoRI fragment of S. typhimurium DNA (positions 9.57 to
20.59) from which the probe was derived (Fig. 2).
All EcoRI fragments from the S. typhi and S. paratyphi A
cosmid clones were subcloned into plasmid vectors pBR325
and pUC9 (Fig. 1). However, only the relevant subclones are
discussed here. The 11.0-kb fragments corresponding to the
S. typhimurium fragment from positions 9.57 to 20.59, which
covers most of the rfb gene cluster, were examined in detail.
Restriction enzyme digests with HindIII, KpnI, ClaI, HpaI,
PstI, XbaI, and BglII gave identical patterns to those given
with the corresponding S. typhimurium fragment. A SacI
site which was absent in S. typhimurium was found in S.
typhi Ty21a and S. paratyphi A IMVS1316 and mapped at a
position corresponding to coordinate 12.6 of S. typhimu-
rium, and a SacI site at coordinate 9.97 in S. typhimurium
LT2 was absent in both S. typhi Ty2la and S. paratyphi A
IMVS1316. We conclude that DNA between the EcoRI sites
at positions 9.57 and 20.59 in S. typhimurium LT2 is essen-
tially homologous with the corresponding regions in S. typhi
Ty2la and S. paratyphi A IMVS1316 DNA. This conclusion
was supported by hybridizing radioactive S. typhimurium
DNA from this region with blotted DNA fragments from
double enzyme digests of S. typhi Ty2la and S. paratyphi A
IMVS1316 DNA from this region.
Restriction site mapping was carried out on the 6.8-, 2.3-,
and 1.8-kb EcoRI fragments from S. typhi Ty2la and the
6.8-, 2.8-, 2.3-, and 1.8-kb EcoRI fragments from S. paraty-
phi A IMVS1316. We determined the order of these frag-
ments by using them separately as probes to hybridize with
double enzyme digests of chromosomal DNA; we then
constructed a restriction map of this region, from positions 0
to 10.9 of S. typhi Ty2la and positions 0 to 16.5 of S.
paratyphi A IMVS1316 (Fig. 3). All restriction sites on the
0
L
9.57
pPR443
I
1
I
nDlhLI.
I
6.8-,
IMVS1316 were found to map at the same positions on the
corresponding fragments of S. typhi Ty2la. The 2.8-kb
EcoRI fragment, which stains with an intensity approxi-
mately twice that expected by comparison with adjacent
bands, appears from its restriction map to duplicate the
adjacent ends of the 6.8- and 1.8-kb S. typhi Ty2la EcoRI
fragments (Fig. 3). We conclude that a 2.8-kb region is
triplicated (Fig. 4). NruI, HpaI, EcoRI, and ClaI digests all
gave a 2.8-kb fragment, which was shown by densitometry
of gel photographs to be present at twice the molarity of
other fragments. This confirmed the 2.8-kb triplication and
showed that one end lies between positions 4.87 and 5.50.
Further confirmation was given by the existence of the
expected
12.1-kb KpnI fragment
(pPR615) which extends from position 2.47 to position 14.55
(Fig. 3).
To identify the region of nonhomology between these
three Salmonella species, we purified a 9.57-kb EcoRI S.
typhimurium DNA fragment from pPR301 (2) for use as a
radioactive probe against EcoRI digests of chromosomal
DNA from S. typhi Ty2la and S. paratyphi A IMVS1316.
The probe, which contained S. typhimurium DNA between
map positions 0 and 9.57, hybridized strongly with the 6.8-kb
and weakly with the 2.3-kb EcoRI fragments from these
strains. In addition to these fragments, the probe hybridized
weakly with the 2.8-kb EcoRI fragment unique to the S.
paratyphi A IMVS1316 digests (Fig. 5). The common 1.8-kb
fragment did not exhibit any detectable homology with the
9.57-kb S. typhimurium DNA probe.
To determine whether the homology extended beyond rfb
toward the his operon, we used plasmid pPR294 (1), which
covers 3.35 kb directly to the left of the EcoRI site at
2.3-, and 1.8-kb fragments from S. paratyphi A
in the chromosome
20.5911L....- S.typhimurium LT2
111U
11 111
pPR45
I
11111
_
pPR446
I
I
I
I
I
II
I
I
pPR47
pPR48 -
I
II
I
II III
11111
11111
pPR49
II
I
I
I
l
pPR430
IAI
I
II IlI
3
I
I
I
11111
pPR450
I
pPR429
/
124
FIG. 1. EcoRI restriction maps of cosmids carrying part of the rfb operQns from S. typhi Ty2la (pPR443, pPR444, pPR445, pPR446,
pPR447, pPR448, pPR449, and pPR450) and S. paratyphiA (pPR430 and pPR429). The approximate extent of therfbregion ofS. typhimurium
LT2 is indicated by a heavy line. The other cosmids are aligned at the conserved EcoRI site at coordinate 9.57 of S. typhimurium LT2. The
numbers 1, 2, 3, and 4 indicate the rfb DNA fragments that were subcloned to form plasmids pPR505, pPR434, pPR436, and pPR437,
respectively.
J. BACTERIOL.
I
I

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O-ANTIGEN VARIATION IN THREE SALMONELLA STRAINS
mn .4
AL.O
O°C7C>0c>
-~~~~~2
thc
cx zcr
CYz
V
yVyVVVVY
FIG.
2.
typhi Ty2la and S. paratyphi A IMVS1316 cosmid
digested
with EcoRI
and probed
radioactive fragment of S. typhimurium LT2rjbDNA (positions
9.95 to 12.20) from pPR300. Lanes 1, 2, and 3 contain S. typhimu-
rium LT2, S. typhi Ty2la, and S. paratyphi A IMVS1316 chromo-
somal EcoRI diges'ts, respectively.
clones
withthe
2.25-kb Kpnl
position 0,
restriction enzyme digests of chromosomal DNA from S.
typhi Ty2la and S. paratyphi A IMVS1316. The restriction
enzyme Sites toward the his operonb for EcoRI, Hpal, Bgll,
and Psti were mapped previously in S.
and found to be conserved in S. typhi Ty2la and S.paratyphi
A IMVS1316.
Thus,
asa molecular probe to hybridize with several
typhimurium (1, 4)
the restriction analyses presented above showed
most of the
sites from positions
position 9.57 rightward on the S. typhimurium rib map were
conserved, with the 9.57-kb EcoRI
corresponding to the EPcoRI
sites at.position 10.9 of S. typhi
Ty21a and position 16.5 of S. paratyphi A IMVS1316. The
junction between the hoinologous and nonhomologous
gions was localized by heteroduplex analysis as follows.
Plasmid pPR505, which consists of a 6.8-kb EcoRi
fragment from S. paratyphi A IMVS1316 (positions 0 to 6.8)
that
0
to
5.5 and from
site of S. typhimurium
re-
DNA
E
S
H H K HpK P C BaC H P Hp
U-
CDour
L 00 1
.
H--NC>iC CN
i((r'
II
r-, 0 UNUL
C>l _3 10 CMU" C L LDO 00
.
.
....
.
cq
CO LALO
r-.
O
CD
!
o
.
J%t
-4
mMN
cloned in pcos2EMBL (16) was digested with EcoRI. Plas-
mid pPR508, which is pGB2 (5) carrying the 9.57-kb EcoRI
fragment (positions 0 to 9.57) from S. typhimurium, was
linearized with Sall. Vectors pGB2 and pcos2EMBL were
used because they exhibit no homology with each other (data
not shown). The digestion products were allowed to hybrid-
ize in a heteroduplex reaction. Denaturation and renatura-
tion of these plasmid DNA fragments gave rise to double-
stranded linear molecules which branched into two single
strands at one end (Fig. 6). By measuring the lengths of
double-stranded segments of these molecules, we found that
the length of homologous DNA was about 5.8
(average of 10 measurements), showing that the region of
homology extends from positions 0 to 5.8 of this strain.
To localize the other end of the nonhomologous region,
plasmid pPR617, which contains the 2.3-kb EcoRI fragment
of S. paratyphi A IMVS1316 cloned in pUC9, and plasmid
pPRS07, which is pGB2 carrying the 9.57-kb fragment of S.
typhimurium in the opposite orientation to that in pPR508,
were used in a similar heteroduplex reaction, since pUC9
also has no homology with pGB2. Plasmids pPR617 and
pPR507 were linearized with BamHI and Sall, respectively
before being mixed in a heteroduplex reaction. The double-
stranded region was measured and found to be about 1.4 ±
0.05 kb (average of eight measurements), showing that the
region of homology extends from position 9.57 to about 8.17
on the S. typhimnurium map (Fig. 7).
+ 0.06 kb
DISCUSSION
Soufthern hybridization and heteroduplex analysis demon-
strated that the rJb gene clusters of S. typhimurium LT2, S.
typhi Ty2la, and S. paratyphiA IMVS1316 share substantial
homology. The LT2 regions from positions 0 to 5.8 and from
8.17 to 9.57 were shown to be homologous by heteroduplex
analysis, and with the exception of one SacI site, all restric-
tion sites examined in the region from 9.57 (the first EcoRI
N Hp
00o
ULA'o
LA
PE P
LO C\J
tooo
.
-
.
..r-
PC
E K P
anC
r.,*
0
to an "
.
.
o
I I. I
I
.
oo o
0: LAOt
C%!LA~ CO
'-4
'-1 -4
H BHp Hp E
-r --oC
000
-0
Co o)Co
aee He-
ooOc
0
"--
..
.
O
LA tDtLDA
UE- m-U)I "D r-
DOJLA
LA
L
Ln
Lo Lo
PPR615
S.PATRA1P1i A IMVS 1316
FIG. 3. Restriction map of rfb DNA of S. typhi Ty2la from positions 0 to 10.9 and S. paratyphi A IMVS1316 from positions 0 to 16.5. S.
typhi Ty2la and S. paratyphi A IMVS1316 have substantial similarity in their restriction maps. The S. paratyphi A IMVS1316 map is drawn
such that sites present between positions 0 and 4.87 and positions 14.2 and 16.5 correspond to sites present between positions 0 and 4.87 and
positions 8.6 and 10.9 on the S. typhi Ty2la map, respectively. The sites found to be triplicated in S. paratyphi A IMVS1316 (see text) are
within a 2.8-kb region which includes ihe 2.5-kb segment from coordinates 5.5 to 8.0, indicated by double lines in the S. paratyphiA
IMVS1316 map. Restriction enzyme sites: E, EcoRI; S, SacI; H, Hindlll; K, KpnI; Hp, HpaI, P, PstI; C, ClaI; Ba, BamHI; N, NruI; B,
BglII. Plasmid pPR615 is pUC19 carrying a 12.1-kb KpnI fragment which extends from positions 2.47 to 14.55 on the S. paratyphi A
IMVS1316 map.
i
I I
)l
I
I
11
11
I
I
I
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I
I
I
I III 11I
_1
I
11
I
I
1.
I
I
I I11
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I
I
I it-I[I.-I I.--
III 11I
11
it
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-11
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105
VOL. 170, 1988
S.
Y23A
T

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106
VERMA ET AL.
0.0
PH
5.8
HB
I
I
9.57
I liIIL
20.59
I
I
I1.TYPMR II IM
H B
I
PH
III
/
',10.9
11
,
I
HB
PH
II
,16.5
I I
4,
I
4
2.8 KB
TRIPLICATION
11 111
lIlIl 1
-JS.TYPHI TY21A
S,PARATYPHI A
IMVS 1316
FIG. 4. Comparative map of rfl operons from Salmonella groups B, D, and A. EcoRI sites are indicated by vertical bars. H, B, and P are
HpaI, BglIl, and PstI recognition sites, respectively. Regions shown by restriction site homology and heteroduplex analysis to be homologous
or unique are indicated as follows: -, common to all; _, S. typhimurium LT2 specific;
A IMVS1316. Note that digestion at any site present only once in a triplicated interval produces a fragment of the same length as this interval
and that the fragment is present at twice the molarity of other fragments generated by this enzyme.
Iz, common to S. typhi Ty2la and S. paratyphi
site known after position 8.17) to 20.59 of S. typhimurium
were conserved in S. typhi Ty2la and S. paratyphi A
IMVS1316 DNA. However, it should be noted that although
heteroduplex analysis has indicated homology in the region
from positions 8.17 to 9.57, some restriction site nonhomol-
ogy was detected. The remaining region of homology right-
ward of position 20.59 has not been examined in the same
detail, but presumably the same high level of homology
exists throughout this conserved region.
The DNA between positions 5.8 and 8.17 in S. typhimu-
rium is replaced by a nonhomologous segment in S. typhi
Ty21a and S. paratyphi A IMVS1316 (Fig. 4). The segment
in S. paratyphi A IMSV1316 differs from that of S. typhi
Ty2la only in having a 2.8-kb fragment triplicated, giving
123 45
6
7
9.57
6.8 -
2.8
22 --
1.8
0
:40
FIG. 5. Southern hybridization analysis of EcoRI digests of S.
typhimurium LT2, S. typhi Ty2la, and S. paratyphi A IMVS1316
chromosomal DNA and of various plasmids by using the EcoRI
fragment ofS. typhimurium LT2 rjb DNA from positions 0 to 9.57 as
a radioactive probe. Lanes: 1, S. typhimurium LT2 chromosomal
DNA; 2, S. typhi Ty2la chromosomal DNA; 3, S. paratyphi A
IMVS1316 chromosomal DNA; 4, pPR505; 5, pPR434; 6, pPR436; 7,
pPR437. The EcoRI subclones in lanes 4 to 7 carry the 6.8-, 2.8-,
2.3-, and 1.8-kb rfblDNA fragments, respectively, from S. paratyphi
A IMVS1316. The two bands of least intensity visible in lane 4
correspond to partial digestion products of pPR505.
rise to 5.6 kb of additional DNA. The extent of the triplica-
tion was established by the presence of a 2.8-kb fragment at
twice the molarity ofother fragments in separate digests with
four different enzymes. One end of the triplicated region
must lie between the ClaI and NruI sites at positions 8.0 and
8.6 in S. paratyphi A IMVS1316, and hence the other end
must lie between positions 4.87 and 5.5. Thus, the 2.8-kb
repeated region includes a short piece of DNA homologous
with S. typhimurium LT2, accounting for the homology of
the 2.8-kb EcoRI fragment with pPR301. Abequose is re-
placed by paratose and tyvelose in the 0 antigens of S.
paratyphi and S. typhi, respectively. Other studies in our
laboratory on S. typhimurium LT2 have shown that the
genes for abequose synthesis (rJbF, rJbG, andrjbH) map in
the general region of the structural difference between the
three strains studied (2), and we are continuing our study of
this variable region to precisely locate the genes involved in
the synthesis of these three sugars.
At this stage, it seems reasonable to conclude that S.
paratyphi A IMVS1316, which is expected to differ from S.
typhi Ty2la in lacking CDP paratose-2-epimerase (15, 20),
nonetheless carries all ofthe DNA present in S. typhi Ty2la,
but that the gene for tyvelose synthesis
inactive in S. paratyphi A IMVS1316. Sasaki and Uchida
(18) showed that a group D strain could mutate to lose the
epimerase and then resemble a group A strain by producing
paratose. Our data show that in at least one instance, a
naturally occumng group A strain arose in this way.
is presumably
m
'v'
FIG. 6. Heteroduplex formed between a 6.8-kb EcoRI fragment
from pPRS05 (S. paratyphi A IMVS1316) and plasmid pPR508 (S.
typhimurium LT2) linearized with Sal. The arrow indicates the end
of the region of homology.
GPR.
GP D
GP A -I
LT2
D
....
.I
.-a - i TrnLrIUM I UM
J. BACTERIOL.

Page 5

O-ANTIGEN VARIATION IN THREE SALMONELLA STRAINS
FIG. 7. Heteroduplex formed between plasmids pPR617 and
pPR507 linearized with BamHI and Sall, respectively. The arrow
indicates the end of the region of homology.
ACKNOWLEDGMENTS
We acknowledge the help of S. Dixon and C. Murray of the
Institute of Medical and Veterinary Science, Adelaide, Australia,
for providing S. paratyphi A strain IMVS1316 and antisera for strain
confirmation. We are thankful to R. Czolij for his technical assis-
tance in electron microscopy work.
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