Download A novel locus of Yersinia enterocolitica serotype O:3 involved in

Molecular Microbiology (1995) 17(3), 575-594
A novel locus of Yersinia enterocofitica serotype 0:3
involved in lipopolysaccharide outer core biosynthesis
Mikael Skurnik, 1. Reija Venho, 1 Paavo Toivanen 2 and
Ayman AI-Hendy 2t
1Turku Centre for Biotechnology and 2Department of
Medical Microbiology, University of Turku, Fin-20520
Turku, Finland.
Summary
Yersinia enterocolitica serotype 0:3 strain 6471/76-c
(YeO3-c) was sensitive to bacteriophage (I)R1-37 when
grown at 37°C but not when grown at 22°C because
of steric hindrance by abundant lipopolysaccharide
(LPS) O-side chain (O-antigen) expressed at 22°C.
The transposon library of YeO3-c was grown at 37°C
and screened for phage ~R1-37-resistant transposon insertion mutants. Three types of mutant were
isolated: (i) phage receptor mutants expressing Oantigen (LPS-smooth), (ii) phage receptor mutants
not expressing O-antigen (LPS-rough), and (iii) LPSsmooth mutants with the phage receptor constitutively sterically blocked. Mutant type (i) was characterized in detail; the transposon insertion inactivates
an operon, named the trs operon. The main findings
based on this mutant are: (i) the trs operon is involved
in the biosynthesis of the LPS outer core in YeO3-c;
the nucleotide sequence of the trs operon revealed
eight novel genes showing similarity to known
polysaccharide biosynthetic genes of various Gramnegative bacteria as well as to capsule biosynthesis
genes of Staphylococcus aureus; (ii) the biosynthesis
of the core of YeO3-c involves at least two genetic loci;
(iii) the trs operon is required for the biosynthesis of
the bacteriophage ~R1-37 receptor structures; (iv)
the homopolymeric O-antigen of YeO3-c is ligated
to the inner core in Y. enterocolitica 0:3; (v) the trs
operon is located between the a d k - h e m H and g a l E gsk gene pairs in the Y. enterocolitica chromosome;
and (vi) the phage ~R1-37 receptor is present in
many but not in all Y. enterocolitica serotypes. The
results also allow us to speculate that the trs operon
is a relic of the ancestral rfb region of Y. enterocolitica
Received 17 January, 1995; revised4 April, 1995; accepted 12 April,
1995. l-Present address. McGill University, Montreal Children Hospital Research Institute, Montreal, Canada. *For correspondence.
E-mail [email protected]. Tel. (21) 633 8035; Fax (21) 633
8000.
© 1995 BlackwellScienceLtd
0:3 carrying genes indispensable for the completion
of the core polysaccharide biosynthesis.
Introduction
Lipopolysaccharide (LPS) molecules are composed of
three distinct parts: lipid A, core oligosaccharide and Oantigen. Lipid A anchors LPS to the outer membrane of
the bacteria. The core oligosaccharide contains about
ten sugar residues, is attached to lipid A and can be
divided into inner and outer cores. O-antigen is a long polymer containing tens of repeat units each containing one to
five sugar residues (Schnaitman and Klena, 1993). Yersinia enterocolitica serotype 0:3 is a human pathogen
causing diarrhoea, enterocolitis, mesenteric lymphadenitis and, as sequelae, erythema nodosum and reactive
arthritis.
The LPS of Y. enterocolitica 0:3 has been studied
chemically and recently also genetically. The O-antigen
of Y. enterocolitica serotype 0:3 is a homopolymer composed of 6-deoxy-L-altrose repeating units, linked together
by 1,2 linkages (Hoffman et aL, 1980; Wartenberg et aL,
1983). Recently, the chemical structure of the inner core
polysaccharide composed of seven sugar residues was
determined (Radziejewska-Lebrecht et aL, 1994) and preliminary structural analysis showed that there are six sugar
residues in the outer core (J. Radziejewska-Lebrecht, M.
Skurnik, A. S. Shashkov, and H. Mayer, unpublished).
The expression of the hornopolymeric O-antigen is temperature regulated such that the number of repeating
units per LPS molecule is higher when bacteria are
grown at 22°C (Acker et aL, 1981; AI-Hendy et al.,
1991b; Kawaoka et al., 1983; Wartenberg et aL, 1983).
Acker and co-workers (Acker et al., 1981) demonstrated
that the enterobacterial common antigen (ECA) of Y.
enterocolitica serotype 0:3 strain was sterically blocked
by abundant O-antigen from ECA-specific antibody molecules in bacteria grown at 22°C but not in bacteria
grown at 37°C. For a number of years we have conducted
studies on the genetics of the lipopolysaccharide biosynthesis and regulation of Y. enterocolitica (AI-Hendy et al.,
1991a,b; 1992; Skurnik and Toivanen, 1993; Zhang et
aL, 1993) and, during these studies, became interested
in the temperature regulation of the O-antigen.
In this work we isolated from sewage a Y. enterocoliticaspecific bacteriophage ~bR1-37 that could only infect a
576
M. Skurnik, R. Venho, P. Toivanen and A. AI-Hendy
serotype 0:3 strain 6471/76-c (YeO3-c) when the bacteria
were grown at 37°C. Thus, the phage behaved analogously to the ECA-specific antibody molecules, indicating
that the phage receptor was sterically blocked by abundant O-antigen in bacteria grown at 22°C. We isolated
three phenotypically different phage ~R1-37-resistant
transposon insertion mutants of YeO3-c and report
detailed characterization of one of them.
Results
Isolation and characterization of bacteriophage
(~R1-37
Bacteriophage (~R1-37 was isolated using a rough Y.
enterocolitica mutant, YeO3-R1, for enrichment, as
described in detail in the Experimental procedures.
YeO3-R1 is a derivative of strain YeO3-c (6471/76-c;
Table 5 later) unable to express the O-antigen because
of a spontaneous mutation in the rfb gene cluster (AIHendy et aL, 1992). Interestingly, the phage could not
infect the smooth parental strain YeO3-c grown at 22°C
but could when the bacteria were grown at 37°C (Table
1). Furthermore, virulence plasmid (pYV)-positive strain
6471/76 (YeO3) was resistant to the phage even when
grown at 37°C (pYV expresses the virulence factors
YadA, which forms surface fibrillae, and Yops, which are
secreted proteins (for a review, see Straley et aL, 1993))
in contrast to YeO3-c, the pYV-cured strain. The rough
virulence plasmid-positive strain YeO3-R2, however, was
sensitive both at 22°C and at 37°C (Table 1). We
reasoned that the phage ¢)R1-37 receptor must be located
close to the outer membrane lipid bilayer and that it would
be sterically blocked both by abundant O-antigen, as seen
with YeO3-c at 22°C, and by less abundant O-antigen plus
abundant YadA/Yops, as seen with YeO3 at 37°C (Table
1). Sensitivity of the rough strain YeO3-R2 at higher temperature indicated that YadA/Yops alone were not sufficient to block the receptor.
Table 1. Effect of growth temperatureon phage sensitivityof
Y. enterocolitica O:3-strains.
Strain
YeO3-c
Description
Smooth,
pYV-negative
YeO3
Smooth,
pYV-positJve
YeO3-R1 Rough,
pYV-negative
YeO3-R2 Rough,
pYV-positive
Growth
temperature
(°C)
22
37
22
37
22
37
22
37
Formation of plaques
with bacteriophage
¢)YEO3-12 ~R1-37
+
+
+
+
-
+
+
+
+
+
Isolation of bacteriophage ~R1-37-resistant
transposon insertion mutants
A transposon library of YeO3-c was constructed as
described in the Experimental procedures. More than
3000 independent mutants were pooled. Bacteriophage
~R1-37 infects only YeO3-c grown at 37°C (Table 1);
therefore, the transposon library was grown at 37°C and
screened for phenotypical phage-resistant mutants. The
mutants either have the phage receptor sterically blocked
through expression of abundant O-antigen or the phage
receptor eliminated by an insertion in the gene(s) responsible for the biosynthesis of the receptor structure. Screening resulted in a number of ~R1-37-resistant mutants,
represented by strains YeO3-RfbR12, YeO3-RfbR14 and
YeO3-RfbR7. Bacteriophage ~YeO3-12 and Moab A6,
both specific for the O-side chain of YeO3, were used to
show that YeO3-RfbR12 and YeO3-RfbR14 expressed
O-antigen and that YeO3-RfbR7 did not (data not shown,
but see Fig. 2 below, lanes 8, 16 and 15, respectively).
Characterization of YeO3-RfbR12
From Southern blots, only a single copy of the transposon
was observed in the chromosome of YeO3-RfbR12 (not
shown). The affected locus was designated trs (for temerature response). To clone the trs locus, two approaches
were followed in parallel to generate a probe for colony
hybridizations. In the first, a genomic library of YeO3RfbR12 was constructed using Pstl-digested chromosomal
DNA from YeO3-RfbR12. This library was screened with
a probe specific for the inverted repeat of TnS, one positive clone containing a 22 kb Pstl fragment was obtained,
and it was used as a probe to screen the YeO3-c genomic
library (AI-Hendy et aL, 1991b). Altogether, 15 positive
clones were isolated from the library. In the second
approach, a modified ligation-polymerase chain reaction
(PCR) method (Rich and Willis, 1990) was used as
described in the Experimental procedures. The purified
ligation-PCR fragments were used as probes to screen
15 positive clones obtained earlier with the 22kb pUC18
Pstl clone; only one of them was positive, carrying a
recombinant plasmid pAM100. The other 14 apparently
carried chromosomal DNA inserts located distant from
the transposon insertion site; therefore, they were discarded. By sequencing out from the transposon using the
22kb pUC18 Pstl clone as template and Tn5-S as
primer, we identified the transposon insertion site; and
the gene carrying the insertion was named trsA. We
continued sequencing with primers based on the new
sequence using pAM100 as a template. With one of the
sequencing primers as an oligonucleotide probe, the genomic library of YeO3-c (see above) was rescreened, and
four additional positive clones were isolated. Restriction
~.~1995 BlackwellScienceLtd, MolecularMicrobiology,17, 575-594
Bacteriophage-resistant LPS mutants of Yersinia enterocolitica 0:3
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M. Skumik, R. Venho, P. Toivanen and A. AI-Hendy
Fig. 2. DOC-PAGE analysisof isolated LPS of YeO3 and derivativestrains. LPS isolatedfrom the Salmonellastrains and from Ye75S,
Ye75R and YeO3-R1 was highly purified (large-scaleisolation)and LPS from the other strains was extractedby the small-scalepurification
method. LPS samples were separated in a 13% separatingand 4% stacking DOC-polyacrylamidegel, 0.35mm thick, and the gel was stained
with silver. Lane 1, Salmonellaminnesota R60 (Ra chemotype); lane 2, SalmonellaminnesotaR5 (Rc chemotype); lane 3, S. minnesota R595
(Re chemotype); lane 4, Y. enterocoliticaYe75S; lane 5, Y. enterocoliticaYe75R; lane 6, YeO3-c; lane 7, YeO3-R1; lane 8, YeO3-RfbR12;
lane 9, YeO3-RfbR12-R; lane 10, YeO3-RfbR12/pRV16;lane 11, YeO3-RfbR12/pRV17; lane 12, YeO3-trsl1; lane 13, YeO3-C-trsS; lane 14,
YeO3-c-trs8-R; lane 15, YeO3-RfbR7; lane 16, YeO3-RfbR14; lane 17, YeO3-RfbR14-R; lane 18, YeO3-RfbR14/pRV16; lane 19, YeO3RfbR14/pRV17. The locationof the O-antigenand the Ra type and the trs type core bands are indicated.The O-antigenlength distribution
corresponds to about 35-70 6-deoxy-L-altroseresiduesper LPS molecule. Ra, Rc and Re chemotypecore oligosaccharidesof the Salmonella
mutants containten, seven and three sugar residues, respectively,and the inner core of Ye75R containseight sugar residues.
maps show that these overlapping clones occupy
altogether about 24 kb of YeO3-c chromosome (Fig. 1).
Construction oftrs mutants
To confirm that the phenotypical changes observed are the
result of a transposon insertion in the trsA gene and not a
second-site mutation, two strategies were followed: directed
mutagenesis and trans-complementation experiments. For
directed mutagenesis, a suicide plasmid, named pRV19GB (kanamycin resistant, Km R, and chloramphenicol
resistant, CImR), was constructed, which carries a KmGenBIock inserted in the 2.9 kb Clal fragment in such a
way that the two internal Nsil fragments were deleted
from the Clal fragment (Fig. 1). pRV19-GB was introduced by mobilization from S m l 0 ~'pir to YeO3 and
YeO3-c. Km R CIm s transconjugants were selected as
described in the Experimental procedures. The proper
insertion of the Km GenBlock into the trs operon of both
strains was confirmed from Southern blots (not shown).
The virulence plasmid-positive derivative was designated
YeO3-trs11, and the virulence plasmid-negative derivative designated YeO3-c-trs8. Phenotypically, these mutants
resembled YeO3-RfbR12, i.e. they expressed O-antigen
(Fig. 2, lanes 12 and 13, respectively) and were resistant
to ~bR1-37 (Table 2). These results ruled out the possibility
of second-site mutations behind the phenotypic changes in
YeO3-RfbR12.
Trans-complementation of the phage-resistant
mutants
Plasmid constructions (pAM200, pRV12, pRV15, pRV16
and pRV17) used for trans-complementation contained
regions from both sides of the transposon insertion site
in YeO3-RfbR12 and are shown in Fig. 1. Transcomplementation of YeO3-RfbR12, YeO3-trs11 and
YeO3-RfbR14 (the last strain was included to see if the
basis of its phage resistance was similar to that of YeO3R12) was monitored using phage ~R1-37 (Table 2). However, trans-complementation was not straightforward. The
largest of the plasmid constructs, pRV16, seemed to transcomplement the mutation in YeO3-RfbR12, but even in this
case, trans-complementation was only postulated; a drop
of a high-titre phage preparation did not produce a clear
hole in the bacterial lawn similar to that occurring with control strains, such as YeO3-c grown at 37°C or YeO3-RI.
Instead, only a slight roughing of the surface of the bacterial lawn was visible. Therefore, the result of this transcomplementation was not conclusive. With YeO3-RfbR14/
pRV16, the roughing was a little more prominent than
with YeO3-RfbR12/pRV16. The inability to trans-complement the phage receptor with any of the constructs was
intriguing; either the genes were not properly expressed
from the plasmids, some important gene(s) were missing
or the presence of the plasmid construct disturbed the
cell surface properties of trans-complemented strains.
The last explanation was most attractive, since slight
roughing was indicative of inefficient infection by the
phage, apparently because of sterical blocking of the
phage receptor in target bacteria.
Search for the phage receptor
Phage-resistant mutants isolated this far were of two types:
those expressing the O-antigen (YeO3-RfbR12, YeO3trs11, YeO3-c-trs8 and YeO3-RfbR14) and those that did
not (YeO3-RfbR7). In the former class, the phage receptor
could be mutated or it could be blocked by abundant Oantigen, while in the latter the phage receptor was apparently mutated since there was no O-antigen to block it.
© 1995 BlackwellScienceLtd, MolecularMicrobiology,17, 575-594
Bacteriophage-resistant LPS mutants of Yersinia enterocolitica 0.3 579
Table I. Bacteriophage ~R1-37 sensitivity of smooth and spontaneous rough derivatives of transposon insertion mutants and
GenBIock mutants with or without trans-complementation.
Trans-
Strain
Description
complementing
plasmid
YeO3-RfbR12
trsA::Tn5-Tcl;
-
~R1-37
sensitivitya
pYV negative
pRV12
pRV15
pRV16
pRV17
pAM200
YeO3-RfbR12-R
(+)
Trans-complementation of rough derivatives
Spontaneous
rough derivative
of YeO3RfbR12
pRV12
pRV15
pRV16
pRV17
pAM200
YeO3-trs 11
YeO3-trsl 1-R
YeO3-c-trs8
YeO3-c-trs8-R
YeO3-RfbR14
YeO3-RfbR14-R
YeO3-RfbR7
+
A trsABC :: km';
pYV positive
pRVl 6
pRV17
(+)
pRV16
pRV17
+
p RV 16
pRV17
(+)
Spontaneous
rough derivative
of YeO3-trsl 1
AtrsABC : : kmr ;
pYV negative
Spontaneous
rough derivative
of YeO3-c-trs8
YeO3-c::TnphoA; pYV
negative
Spontaneous
rough derivative
of YeO3RfbR14
(if present) and make it available for the phage. To test
this we used phage ~YeO3-12 to isolate O-antigennegative derivatives of the mutants. The rough derivatives
were tested with ~R1-37 (Table 2). The results revealed
that rough trs mutant strains were still resistant to the
phage while YeO3-RfbR14-R was clearly sensitive. These
observations indicated that in the trs mutants the phage
receptor is mutated while in YeO3-RfbR14 it is sterically
blocked.
--
+
pRV16
pRV17
+
YeO3-c::Tn-phoA
a. (+), incomplete trans-complementation, see text for details.
Southern hybridizations (not shown) using pRY5 and
pRV7 (Fig. 1) as probes revealed that transposon insertion in YeO3-RfbR14 was not located within the 24 kb trs
region spanned by the probes. This indicated that the
genetic basis of the phage resistance of YeO3-RfbR14
was distinct from that of YeO3-RfbR12.
To analyse whether the phage receptor was constitutively sterically masked in the mutants, we reasoned that
mutants unable to produce any O-antigen would become
phage sensitive again provided that the phage receptor
was not mutated. The identity of the phage receptor should
not play any role in rough derivatives of the mutants: in
any case, lack of O-antigen would unmask the receptor
© 1995 Blackwell Science Ltd, Molecular Microbiology, 17, 575-594
With this new information available, we wondered whether
we could see trans-complementation of phage ~R1-37
receptor by the trs locus in rough derivatives of the
mutants. To test this, rough derivatives of previously
constructed trans-complementation strains were selected
using phage ~YeO3-12 as above and tested for phage
~R1-37 sensitivity (Table 2). Plasmids pAM200 and
pRV12 did not trans-complement the mutation. The latter
contains 1.4 kb upstream and 5.2 kb downstream of the
transposon insertion site. This was also the case with
pRV15, which contains the trsA gene under the control
of the Ptac-promoter. pRV16 was the only plasmid able
to trans-complement phage receptor in rough derivatives.
The phage droplet formed a clear hole in the bacterial
lawns of rough derivatives (in contrast to those of smooth
derivatives) containing pRV16, indicating a complete
lysis and unquestionable trans-complementation, pRV16
contains about 12kb of YeO3 DNA, localized 1.4kb
upstream and 10.6kb downstream of the transposon
insertion site. The insert in pRV16 contains a single Nrul
site about 8 kb downstream of the transposon insertion
site. This allowed construction of pRV17, which also
was unable to trans-complement the mutation in YeO3RfbR12. In conclusion, these experiments clearly demonstrate that structural requirements of phage ~R1-37 receptor are determined by the trs locus and that the locus is an
operon longer than 8 kb in size. Strangely, YeO3-RfbR14R/pRV17 was resistant, while both YeO3-RfbR14-R and
YeO3-RfbR14-R/pRV16 were sensitive.
LPS profiles of ~R1-37-resistant mutants
The above results suggest that phage ~bR1-37 receptor
could be a structural component of the LPS molecule.
LPS was isolated from the different strains obtained
during this work and deoxycholate (DOC)-PAGE analysis indeed revealed major differences in LPS profiles of
~R1-37-resistant mutants. Wild-type LPS of YeO3-c
(Fig. 2, lane 6) contains two populations of LPS molecules. The population of LPS molecules composed of
all three structural blocks: lipid A, core and O-antigen
(the last with varying numbers of repeating units), forms
580
M. Skurnik, R. Venho, P. Toivanen and A. AI-Hendy
a broad band at the top of the gel. The second population
of LPS molecules missing the O-antigen (Ra type LPS)
forms a band in the lower part of the gel. This band is
shown best by the single band formed by LPS isolated
from YeO3-R1 (Fig. 2, lane 7). LPS isolated from mutants
YeO3-RfbR12, YeO3-trsl I and YeO3-c-trs8 (Fig. 2, lanes
8, 12, and 13, respectively) formed the O-antigen band
similar to the wild-type LPS but with a smaller sized (trstype) core band. We included in the analysis as controls
LPS prepared from Y. enterocolitica Ye75S (a German
serotype 0:3 isolate) and Ye75R, which is a rough
UDP-4-epimerase-deficient derivative of Ye75S that is
defective in the synthesis of the outer core of LPS (Radziejewska-Lebrecht et aL, 1994). The migration of the trstype core band and the core band of Ye75R are almost
identical (Fig. 2, lane 5), although in the latter a small portion of the LPS appeared to have complete core. YeO3RfbR7 (Fig. 2, lane 15) did not possess O-antigen and
its LPS had a trs-type core. YeO3-RfbR14 (Fig. 2, lane
16), however, had a wild-type LPS profile indistinguishable from that of YeO3-c (Fig. 2, lane 6). Core-band
mobility shifts were also apparent in rough derivatives of
the trs mutant strains (Fig. 2, lanes 9 and 14). LPS patterns of YeO3-RfbR12/pRV16 (Fig. 2, lane 10) and
YeO3-trs11/pRV16 (not shown) were close to the wildtype pattern showing that trans-complementation almost
completely corrected the outer-core defect. Presence of
pRV16 in YeO3-RfbR14 (Fig. 2, lane 18) did not affect
the LPS profile of this strain; however, the presence of
pRV17 in YeO3-RfbR14 (Fig. 2, lane 19) dramatically
affected the LPS such that most of its core changed to
the trs type. This change is in line with the disappearance
of phage ~R1-37 receptor from this strain (Table 2).
These results suggest that the function of the trs operon
is the synthesis of the LPS outer core structure, and also
that the complete core might be the phage receptor.
However, if the (outer) core was the phage ~R1-37
receptor, then purified LPS from YeO3-R1 (Fig. 2, lane
7) should be able to inhibit the phage. This was indeed
the case, but the concentration needed for inhibition was
so high (>10 mg m1-1, not shown) that this result was not
convincing.
Distribution of phage #)R1-37 receptor in genus
Yersinia
The presence of phage ~R1-37 receptor structure in
various Yersinia species was tested by analysing the sensitivity profiles of bacteria to phage ~R1-37. The bacteriophage receptor was present, as judged by the ability of
phage ~R1-37 to infect and lyse target bacteria, in many
but not all Y. enterocolitica strains representing 13 different serotypes (Table 3). The majority of Y. enterocolitica
serotypes were resistant to ~R1-37. The presence or
absence of phage receptor did not correlate with the pathogenic potential of the different Y. enterocolitica serotypes.
Five serotypes contained both sensitive and resistant
strains. Although we did not attempt to elucidate the
reason behind the heterogeneity of strains within the five
serotypes, one could speculate that resistance could be
caused by steric hindrance of the phage by YadNYops
or by a capsule elaborated by the bacteria and not
through the absence of the receptor structure itself.
Altogether, the phage receptor was present in about one
third of the tested serotypes (Table 3). Within other Yersinia species, only one Y. intermedia strain was sensitive
to (~R1-37 (Table 3).
Nucleotide sequence of the trs locus
The 13.6 kb nucleotide sequence over the trs region was
determined. A summary of the sequence information is
Table 3. Bacteriophage ~R1-37 sensitivity of Yersinia species (number of studied strains given in parenthesis).
Yersinia species
(~R1-37
sensitive
serotypes
Y. enterocolitica
O:1 (2)
0:3 (5)
0:6 (1)
O:6,31 (1)
0:9 (7)
0:25,26,44 (1)
O:41,43 (1)
0:5o (1)
Y.
Y.
Y.
Y.
Y.
Y.
Y.
pseudotuberculosis
intermedia
kristensenii
frederiksenii
mollaretii
bercovieri
ruckerii
0:52,54
Serotypes with
(~R1-37 sensitive
(+) and resistant
( - ) strains
0:2 (1+/1 - )
0:5,27 (3+/1-)
0:5 (5+/1 - )
O:21 (1+/2-)
O:41 (27)43 (1+/1 - )
~R1-37-resistant serotypes
O:1,2,3 (1)
0:6,30 (3)
O:10 (4)
O:13a,13b (1)
O:15 (1)
0:26,44 (1)
0:35,36 (1)
O:41(27)K1 (1)
0:4 (1)
0:4,32 (1)
0:7,8 (3)
0:8 (6)
O:13 (1)
O:13,7 (2)
O:13,18 (1)
O:14 (1)
0:20 (2)
0:25 (1)
0:28,50 (1)
0:34 (1)
0:35,42 (1)
O:41(27),42 (1)
K1 non-typable (2)
non-typable (3)
i, iA, ill (4)
0:16,21 (1)
0:12,25, O:16, non-typable (5)
O:16, 0:35, 0:48, O:58,16, K1, non-typable (6)
0:59(20,36,7) (1)
O:58,16 (2)
(1 strain)
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M. Skurnik, R. Venho, P. Toivanen and A. AI-Hendy
Table 4. trs operon-encoded proteins: properties and similarity to sequences in the databases.
YeO3
Size
polypeptide (aa/kDa)a
pl
Similar polypeptides (abbreviations inside parenthesis refer to
the abbreviations used in Figs 3-6)
Database accession no.
Degree of
identity (%)
Adk
214/23.8
6.69
E. coli Adk, adenylate kinase
D90259, X03038, X57315
> 80
HemH
308/34.9
7.42
E. coli: VisA/HemH, ferrochelatase
D90259, L01777
> 60
TrsA
418/47.2
10.15
E. coli RfbX, putative O-side chain translocase
Shigella flexneri, S. dysenteriae RfbX
Salmonella enterica, S. typhimurium RfbX
Haemophilus influenzae: Isg-locus, ORF1
U03041 U09876
X71970, L07293
M65054, X56793
M94855
23-28
TrsB
TrsC
318/37,1
292/33,8
M31722
X06690 U00039
M94855, Z37516
17-25
7.97 Anabaena, unknown ORF (Anaheta)
9.01 E. coli, YibD, close to rfa gene cluster (YibD)
H. influenzae: Isg-locus ORF5 (Hinf), capsulation locus ORF
(Hinf3)
Vibrio anguillarum virulence protein (Vang)
Y. enterocolitica RfbB (Ye RfbB)
Neisseria gonorrhoeae glycosyltransferases (Gc2, Gc5)
Rhizobium meliloti glycosyltransferases (ExoO, ExoU, ExoM,
ExoW)
Bacillus subtilis ipa-63d and ipa-56d gene products (Bs56,
Bs63)
S. flexneri dTDP-rhamnosyl transferase (RfbG)
Plant symbiont bacteria NodC, N-acetylglucosaminyl
transferase, (NodCm, NodCp, NodCa)
Erwinia amylovora exopolysaccharide synthesis (AmsE,
AmsB)
Yeast dolichylphosphate glucosyltransferase (Scerl)
Yeast dolichylphosphate mannosyltransferase (Dpml)
Salmonella typhi ORF (Ty2)
S. typhimurium ORF in the rfb region (Yrf9),
rhamnosyltransferase (RfbX)
Salmonella choleraesuis ORFs (Schl, Sch2)
Streptococcus pyogenes hyaluronan synthase (Hy.~ll)
Stigmatella aurantiaca FBFA protein (Stig)
Anacystis nidulans CobA (Anal)
TrsD
TrsE
358/40.8
344/38.1
10.24
7.42
TrsH
349/39.8
8.03
TrsF
341/37.6
8.22
L08012
Z18920
U14554
Z22636, L20758
X73124
X71970
X01649, P24151, Q07755
X77921
X77573
P14020
B42476
X56793, P26403
$22615, $22617
JC2077
$18962
X70966
H. influenzae: Isg-locus ORF3 (H. inf.)
Erwinia amylovora AmsD, exopolysaccharide synthesis
(AmsD)
E. coli: RfaB (RfaB)
B. subtilis: RodD (RodD)
Saccharomyce$ cerevisiae SPT14 transcription factor
(SPT14)
Mycobacterium leprae ORF (M. lep.)
Maize sucrose synthase, UDP-glucose-fructose-phosphate
glucosyltransferase (Sps)
S. typhimurium RfaK, N-acetyiglucosaminyltransferase (RfaK)
Human N-acetylglucosylphosphatidylinositol biosynthetic
protein (Piga)
E. amylovora AmsK (AmsK)
S. enterica ORF in rfb region (Yrfl)
E. coli MtfA, mannosyltransferase (MtfA)
Klebsiella pneumoniae RfbC, galactosyltransferase (Kpn)
Serratia marcescens RfbF, galactosyltransferase (RfbF)
Staphylococcus aureus capsulation proteins (CapM, Capri)
Vibrio cholerae putative outer membrane protein (Vc ORF)
S. typhi VipC, Vi capsule biosynthesis M94855
M94855
X77921
Pseudomonas aeruginosa RfbA
B. subtilis MraY, phospho-N-acetylmuramoyl-pentapeptide
transferase (Bs MraY)
E. coil MraY, phospho-N-acetylmuramoyl-pentapeptide
transferase (Ec MraY)
S. aureus locus involved in high methicillin resistance (S.
aureus)
Sulfolobus acidocaldarius UDP-N-acetylglucosamine-dolichylphosphate N-acetylglucosaminophosphotransferase (Sulfol.
Nac)
U17293
Z15056, Q03521
38 (TrsD)
34 (TrsD)
$39643
X15200
X63290
U00018
P31927
P26470
P37287
16-26
X77921
X56793
D43637
L31762
L34167
U10927
$28485, X59554
D14156
54
P15876
D21131
B54058
23-29
© 1995 Blackwell Science Ltd, Molecular Microbiology, 17, 575-594
Bacteriophage-resistant LPS mutants of Yersinia enterocolitica 0.3 583
Table 4. Continued.
Ye03
Size
polypeptide (aa/kDa)a
pl
Similar polypeptides (abbreviations inside parenthesis refer to
the abbreviations used in Figs 3-6)
E. coil Rfe, UDP-N-acetylglucosaminotransferase (Rfe)
Mouse UDP-N-acetylgiucosamine-dolichyl-phosphate Nacetylglucosaminophosphotransferase (Mouse Nac)
Streptomyces coelicolor unknown protein (Streptom.)
TrsG
638/70.4
8.00
V. cholerae: unknown partial ORF downstream of the rfb
gene cluster (Vc ORF)
S. aureus CapD, type 1 capsular polysaccharide biosynthesis
(CapD)
Acinetobacter sp. acetoacetyI-CoA-reductase (Accp)
Sus scrofa 17-beta-oestradiol dehydrogenase (Ss17)
Neisseria meningitidis TDP-glucose dehydratase (Gdhnm)
E. coil RifE, UDP-N-acetylglucosamine epimerase (Rife)
Yeast TDP-glucose-4,6-dehydratase (Sere)
Streptomyces fradiae dTDP-glucose dehydratase (Sfu)
Streptomyces griseus dTDP-glucose-4,6-dehydratase (Stre)
Pachysolen tannophilus GalE, UDP-glucose-4-epimerase
(GalEpt)
Kluyveromyces marxianus GalE, UDP-glucose-4-epimerase
(GalXk)
Consensus sequence of 8 bacterial GalEs, UDP-glucose-4epimerase (GalE cons)
Azospirillum brasiliense GalE, UDP-glucose-4-epimerase
Database accession no,
Degree of
identity (%)
$30678
$24326
$41945
X59554
64
U10927
43
L37761
X78201
L09188
P27830
L37354
U08223
P29782
$26921
15-25
P09609
$36409
(Azo)
GalE
336/37.1
Gsk
266 N-terminal
6.79
R. meliloti ExoZ, UDP-glucose-4-epimerase (ExoZ)
S. cerevisiae UDP-glucose-4-epimerase (GalX)
S. typhimurium RfbG, CDP-glucose 4,6-dehydratase (RfbG)
B. subtilis ipa-73d gene product (Bsg)
Japanese firefly luciferin 4-monooxygenase, luciferase (Lucl)
X58126
P04397
P26397
X73124
Q01158
GalE, UDP-glucose-4-epimerase
See above
55-65
E. coil Gsk, inosine-guanosine kinase
D00798
75
a. aa, amino acids.
presented in Fig. 1. The sequence revealed that the transposon was inserted in the first gene (trsA) of the trs operon
between nucleotides 2336 and 2337 of the determined
nucleotide sequence. Upstream of trsA was a non-coding
region of 400bp, preceded by two genes highly homologous to the adkand hemHgenes of E. coliand other bacteria. Downstream of trsA, seven new open reading frames
(ORFs) were detected, designated trsBCDEFGH. Downstream of trsH, two genes highly homologous to galE
and gsk were found. Each of the 11 recognized genes is
preceded by a ribosomal binding site motif 3 - 1 0 nucleotides upstream from the start codon of the gene, except
gskfor which no clear motif is present. Weak putative promoter motifs were recognized along the sequence (Harr et
aL, 1985) and one of them is located upstream of the trsA
gene. Successful trans-complementation with plasmid
pRV16 but not with pRV12 implies that there is a functional promoter upstream from the trs operon but apparently not within it. In the non-coding 400bp region
between hemH and trsA, the 39 bp nucleotide sequence
characteristic for the JUMPstart sequence was recognized (Hobbs and Reeves, 1994).
© 1995 Blackwell Science Ltd, Molecular Microbiology, 17, 575-594
Characteristics of the Trs proteins
Table 4 summarizes the properties of the deduced polypeptides and their similarities to sequences in the databases.
TrsA is highly hydrophobic throughout its length, suggesting that it is a membrane-associated protein, apparently spanning the membrane several times; by similarity,
it belongs to the RfbX family of proteins (Table 4, Klena
and Schnaitman, 1993; Reeves, 1993; Schnaitman and
Klena, 1993; Yao and Valvano, 1994). The Haemophilus
influenzae Isg locus ORF1 protein was identified as a
new member of this protein family (Table 4). Alignment
and analysis of similarities of the RfbX family of proteins
including TrsA was performed earlier (Yao and Valvano,
1994). The implications of these findings for the putative
role of TrsA are presented in the Discussion.
TrsB and TrsC are overall hydrophilic proteins and comparison of these proteins with the database identified
about 30 proteins that showed limited similarity (17-25%
identity) to both. Many of these proteins are glycosyl
transferases, both of prokaryotic and eukaryotic origin.
These proteins were aligned and part of the alignment is
shown in Fig. 3A. Outside this region the proteins were
M. Skurnik, R. Venho, P. Toivanen and A. AI-Hendy
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~ 0
"~"
88
©
1995
Blackwell
Science
Ltd,
Molecular Microbiology, 1 7 ,
575-594
Bacteriophage-resistant LPS mutants of Yersinia enterocolitica 0:3
significantly less similar and no consensus sequence was
generated. Inside the region, however, the proteins aligned
better and generated several highly significant local alignments, the most conserved of which are shown in Fig. 3B.
These local alignments for these transferases revealed
consensus motifs that probably interact with either the substrate sugar or the enzyme co-factor during the enzymatic
reaction. Similar local motifs shared by a number of transferases were recently identified by R. Morona (personal
communication, Morona et aL, 1995). Furthermore, hydrophobic cluster analysis identified similar domains in a
number of 13-glycosyl transferases, most of which are
included in the present comparison (Saxena et al.,
1995). The local alignments identified in Fig. 3B match
very well with the identified domains. Therefore, it may
be speculated that TrsB and TrsC both function as I~gly(u)cosyl transferases.
TrsD, TrsE and TrsH are also overall hydrophilic proteins, and the database search identified again a set of
proteins involved in polysaccharide biosynthetic pathways (Table 4). Alignment of these proteins (Fig. 4A)
showed that two of the proteins have significant similarity
to TrsD, the H. influenzae ORF3 in the Isg locus (38% identity) and the Erwinia amylovora AmsD (34% identity). The
other proteins were 16-26% identical with TrsD, TrsE and
TrsH. Also with these proteins, highly significant local
alignments were generated revealing consensus motifs
probably important for the enzymatic function (Fig. 4B).
The E. amylovora AmsD protein is involved in the exopolysaccharide amylovoran biosynthesis (Coplin et al.,
1994). The structure of the amylovoran subunit is proposed to contain galactose and glucuronic acid in a
molar ratio of 4:1, which is decorated with pyruvate and
acetate groups (Smith et aL, 1990). Since the outer core
of 0:3 LPS contains galactose, N-acetylgalactosamine
and glucose but apparently not glucuronic acid (see Discussion), the similarity of TrsD and AmsD suggests that
these proteins are involved in galactose metabolism.
TrsE and TrsH did not show any overall similarity to
known proteins, but together with TrsD they shared a
motif common to galactosyl- and mannosyltransferases,
indicating that these three proteins function as galactosyl- or N-acetylgalactosaminyltransferases.
TrsF was, like TrsA, highly hydrophobic, and the database search identified about ten proteins all again involved
in polysaccharide biosynthetic pathways (Table 4). The
RfbA protein of Pseudomonas aeruginosa serotype 05,
which is involved in the 0 5 O-antigen biosynthesis, was
54% identical with TrsF (Dasgupta and Lam, 1995). Comparable similarities were found for the corresponding protein of P. aeruginosa serotype O11 (J. Goldberg, personal
communication); other proteins were found to be less similar (23-29%). The similarity of all of these proteins was
spread over the entire lengths of these sequences
© 1995BlackwellScienceLtd, MolecularMicrobiology,17, 575-594
587
(Fig. 5). The similarity of TrsF to the RfbA protein of P.
aeruginosa 0 5 and O11 indicates that these proteins
must have similar functions in both organisms. The Oantigens of P. aeruginosa 0 5 and O l l both contain
N-acetylfucosamine, which is a 6-deoxy derivative of Nacetylgalactosamine. The other less similar proteins are
transferases involved in N-acetylglucosamine metabolism. Therefore, it is highly likely that TrsF functions as
an N-acetylgalactosaminyltransferase.
TrsG has a highly hydrophobic N-terminus and a
hydrophilic C-terminus. Comparison with the database
revealed two sets of proteins showing different levels of
similarity (Table 4). Highly similar were the CapD protein
of S. aureus (43% overall identity, C-termini about 60%
identical), involved in the S. aureus type 1 capsular polysaccharide biosynthesis, and the partial ORF of Vibrio
cholerae O1 downstream from the rfb gene cluster (64%
identity, Fig. 6A). A third highly similar protein is encoded
by the downstream gene of the trsFhomologue of P. aeruginosa serotype O l l (not shown, J. Goldberg, personal
communication). More limited similarity (15-25% identity)
was found with a set of proteins involved in sugar (specifically galactose) biosynthetic pathways. Local alignments
(Fig. 6B) revealed again consensus motifs apparently
central for enzymatic function. The S. aureus type 1 capsule is composed of taurine, N-acetylfucosamine and 2acetamido-2-deoxy-D-galacturonic acid (Lin et aL, 1994);
however, no precise function for CapD is known. High similarity between TrsG, P. aeruginosa O11 protein and CapD,
and the role of the proteins in LPS or capsule biosynthesis
suggests a similar function for all including the ORF in V.
cholerae. Based on the presence of N-acetylgalactosamine in the outer core of Y. enterocolitica 0:3 (see the
Discussion), the most likely function of these proteins is
either in biosynthesis of N-acetylgalactosamine (or Nacetylfucosamine) or as a corresponding transferase.
The latter function is more unlikely since none of the
other proteins showing similarity to TrsG is a transferase.
Whatever the role of TrsG, its role in core biosynthesis
was highlighted in YeO3-RfbR14 carrying pRV17. Apparently, the expression of truncated TrsG missing the 98
C-terminal amino acids (Fig. 6A) by the multicopy plasmid has severely disturbed core biosynthesis in YeO3RfbR14, since both the core structure and the phage
receptor were influenced dramatically (Fig. 2, lane 19).
This, of course, also indicates that the C-terminus of
TrsG is crucial for its proper function.
Discussion
A new chromosomal locus involved in the LPS biosynthesis of Y. enterocofitica serotype 0:3 was identified
and characterized in this study. The locus was inactivated
by T n 5 - T c l insertion in strain YeO3-RfbR12. Two
588
M. Skurnik, R. Venho, P. Toivanen and A. AI-Hendy
phenotypical changes took place: (i) the phage ~R1-37
receptor was eliminated, and (ii) the LPS core was
changed. The main conclusions in this study are (i) the
trs operon is involved in biosynthesis of the LPS outer
core in Y. enterocolitica 0:3, (ii) biosynthesis of the core
of Y. enterocolitica 0:3 involves at least two genetic loci,
(iii) the trs operon determines the structures required of
bacteriophage ~R1-37 receptor, (iv) homopolymeric Oantigen of Y. enterocolitica 0:3 is ligated to the inner
core structure, (v) the trs operon is located between the
adk-hemH and galE-gsk gene pairs in the Y. enterocolitica chromosome, and (vi) phage qbR1-37 receptor is present in many but not in all Y. enterocolitica serotypes.
We showed that the trs operon is involved in completion
of the core polysaccharide of YeO3 by directed mutagenesis and trans-comptementation experiments. Transcomplementation experiments using pRV16 gave inconclusive
results with smooth trs mutants. Analysis of their rough
derivatives revealed that the phage receptor was indeed
trans-complemented in smooth strains, but, it was somehow sterically blocked. Preliminary Northern blotting
analysis supports this line of thinking; transcription of the
rfb gene cluster was derepressed at 37°C in these transcomplemented mutants (M. Skurnik, unpublished). Apparently, expression of the trs operon from a multicopy
plasmid disturbs by an unknown mechanism the temperature regulation of the rfb gene cluster of YeO3. Further
work is being carried out by us to elucidate the functions
of the trs operon and its role in regulation of the Oantigen, if any.
We earlier cloned the rfa gene cluster of YeO3 (AIHendy et aL, 1991a): the recombinant plasmid pAN100
carries the rfa gene cluster and expresses in E. coli a
core structure recognized by the YeO3 core-specific
Moab 2B5. Comparison of the restriction endonuclease
maps of the insert in pAN100 with that of the trs operon
showed no similarities, indicating that these are two separate loci. Apparently, biosynthesis of the core of YeO3 is
complicated and involves at least two genetic loci, the rfa
gene cluster and the trs operon for the biosynthesis of
inner and outer core structures, respectively. Further
work is needed to elucidate this question.
We showed that the trs operon determines the structures required for the phage ~R1-37 receptor. Cloned
into pRV16, the trs operon was able to transcomplement
the receptor back into the mutants: moreover, the phage
receptor phenotype and the LPS structure changed hand
in hand. This is indirect evidence suggesting that the
phage receptor is part of the LPS core. We attempted to
inhibit the phage with highly purified LPS from YeO3-R1
but did not obtain convincing results. This may indicate
that the phage receptor is not the LPS core itself, instead
it may be one of the Trs proteins. Since the inhibition
experiment was performed with solubilized LPS, the
conformation of the phage receptor in the LPS may not
be correct under these conditions. Presence of the phage
qbR1-37 receptor in various other Y. enterocolitica serotypes and in Y. intermedia suggests that these strains
possess a similar structure on the surface, and this may
help the future identification of the phage receptor.
The size of the complete (Ra type) core of YeO3
appears to be significantly larger than the Ra type of Salmonella, as shown by the DOC-PAGE analysis (Fig. 2,
lanes 1 and 7), while the truncated core in the trs mutants
migrated between the Ra- and Rc-type LPS of Salmonella
(Fig. 2, lane 2). This suggests that the trs operon is
involved in adding several sugar residues to the core structure of YeO3. Indeed, a preliminary structural analysis of
the complete core of YeO3-R1 showed that there are six
sugar residues in the outer core, which is branched and
composed of glucose, galactose and N-acetylgalactosamine (J. Radziejewska-Lebrecht, M. Skurnik, A. S.
Shashkov and H. Mayer, unpublished). In DOC-PAGE
analysis, LPS from Ye75R migrated almost identically to
the core of the trs mutants, suggesting that Ye75R is
also a trs mutant, although the presence of minor amounts
of complete core in Ye75R indicates that its defect is a little
leaky (Fig. 2, lane 5). Since the trs mutants are able to
express the O-antigen properly, it is likely that the homopolymeric O-antigen is attached to a residue in the inner
core region. Figure 7 shows the hypothetical structure of
the Y. enterocolitica serotype 0:3 LPS based on the findings in this work and in Radziejewska-Lebrecht et aL
(1994) and Radziejewska-Lebrecht et aL (unpublished).
Nucleotide sequence information showed that the trs
operon has some similarities with the rfb gene clusters of
other Enterobacteriaceae. This is a little surprising since
the rfb genes are responsible for the O-antigen biosynthesis. In all known rfb cluster, a gene for a TrsA homologue,
rfbX, is present (Klena and Schnaitman, 1993; Reeves,
1993; Schnaitman et aL, 1993; Yao and Valvano, 1994).
The RfbX protein is thought to be involved in translocating
the cytoplasmically synthesized acyl carrier-bound Oantigen subunit to the periplasmic space, where it is
transferred by the O-antigen polymerase to the growing
O-antigen (Klena et aL, 1993; Reeves, 1993; Schnaitman
and Klena, 1993; Yao et aL, 1994). Another similarity is
that in Y. pseudotuberculosis and Y. enterocolitica serotype 0:8, the rfb gene clusters are located downstream
of the adk and visA/hemH genes (Kessler et aL, 1993; L.
Zhang, P. Toivanen, and M. Skurnik, submitted). Furthermore, in serotype 0:8, the galEand gskgenes were identified downstream of the rfb region (L. Zhang, P. Toivanen,
and M. Skurnik, submitted). Based on these similarities, on
the capsule-like structure of the serotype 0:3 homopolymeric O-antigen and its uncommon linkage to the inner
core, we hypothesize that the trs operon is an rfb relic in
YeO3 and that the rfb region of YeO3 directing the
~) 1995BlackwellScienceLtd, MolecularMicrobiology,17, 575-594
Bacteriophage-resistant LPS mutants of Yersinia enterocolitica 0 : 3
Fig. 7. Hypotheticalschematic structureof Y.
enterocolitica serotype 0:3 LPS. Preliminary
data of the structureof the outer core are
given as sugars 1-6 (Radziejewska-Lebrecht,
M Skurnik, A. S. Shashkovand H. Mayer,
unpublished).The direction of the glycosidic
linkages is indicatedby arrows between the
residues. Defect in trs mutants and in Ye75R
(Rc chemotype mutant) is indicatedby a
dashed line betweenthe outer and inner
cores. The level at which the homopolymeric
O-antigen is attached to the inner core is
indicated. The exact site and bondingare not
known. Abbreviations:LD-hep, L-glycero-Dmanno-heptose; DD-hep, D-glycero-g-mannoheptose; KDO, 3-deoxy-D-manno-octulosonic
acid; D-glc, D-glucose;GIcN, glucosamine.
Outer core
<
<
~rs[
Ye75RF ",,, ~,
<
O-antigen-)
589
Inner core
binding level
LipldA
>
>
>
>
>
>
biosynthesis of the homopolymeric capsule-like O-antigen
(AI-Hendy et aL, 1991b; Zhang et aL, 1993) has taken
over the O-antigen expressing role from the ancestral trs
operon. That the present form of trs operon has been preserved indicates that its role in the completion of the core
structure is indispensable. Preservation of the trsA gene
might indicate that the outer core of YeO3 is synthesized
on an acyl-carrier lipid in the cytoplasmic side of the
inner membrane and then translocated 'en block' to the
periplasmic space by the TrsA protein. In the periplasmic
space, the block is transferred onto the inner core. More
work is needed to elucidate this hypothesis.
The possible roles of other individual Trs proteins can be
considered based on the similarity searches, alignments
shown in Table 4 and Figs 3 - 6 and the structural components of the outer core. Six of the Trs proteins have some
similarities with different glycosyltransferases (TrsB, C, D,
E, F and H). Since this is also the number of sugar residues
in the outer core (Fig. 7), each residue apparently has
its own transferase encoded by the trs operon. For TrsG,
a function in the biosynthesis of N-acetylgalactosamine
is proposed. GalE is the key enzyme in galactose
© 1995 BlackwellScienceLtd, Molecular Microbiology, 17, 575-594
metabolism since it catalyses the epimerization of UDPglucose to UDP-galactose. Since galactose and N-acetylgalactosamine are components of the outer core of YeO3
and the galEgene is located downstream of the trs operon,
it is highly likely that galE belongs to the trs operon.
The phenotype of YeO3-RfbR7 is very close to that of
the rough derivatives of the trs mutants, i.e., it has truncated core, it lacks the phage receptor and it is O-antigen
negative. We have not yet characterized this strain in
detail; presumably it is a trs mutant that has spontaneously lost the O-antigen. The phenotype of YeO3-RfbR14
is distinct from trs mutants and from YeO3-RfbR7. YeO3RfbR14 has wild-type LPS with the complete core and
O-antigen. It is phenotypically phage resistant when grown
at 37°C, in contrast to YeO3-c from which it is derived.
Elimination of the O-antigen from YeO3-RfbR14 revealed
that the strain expresses the phage receptor. These
results indicate that in YeO3-RfbR14 the phage receptor
is sterically blocked both at 22°C and 37°C. The genetic
basis of this blockage is not yet known, presumably the
expression of O-antigen at 37°C is derepressed. We are
presently working on this question.
590
M. Skurnik, R. Venho, P. Toivanen a n d A. AI-Hendy
Table 5. Bacterial strains, plasmids and bacteriophages.
Bacterial strains/Plasmids/
Bacteriophages
Description
Source/Reference
Serotype 0:3, pYV* patient isolate
p Y V - derivative of 6471/76
Spontaneous rough derivative of YeO3-c
Spontaneous rough derivative of YeO3
YeO3-c trsA::Tn5-Tcl; Tet R
Spontaneous rough derivative of YeO3-RfbR12; Tet n
YeO3-c::Tn5-phoA; Km R
Spontaneous rough derivative of YeO3-RfbR14; Km R
YeO3-c::Tn5-phoA; Km R
YeO3-c, AtrsABC::km-GenBIock; Km R
Spontaneous rough derivative of YeO3-c-trs8; Km R
YeO3, AtrsABC::km-GenBIock; Km R
Spontaneous rough derivative of YeO3-trsl 1 ;Km R
Serotype 0:3, German isolate
Spontaneous rough derivative of Ye75S, 4-epimerase deficient
Skurnik (1984)
Skurnik (1984)
AI-Hendy et aL (1992)
AI-Hendy et aL (1992)
This work
This work
This work
This work
This work
This work
This work
This work
This work
Acker et aL (1980)
Radziewskaja-Lebrecht et aL (1994)
thi thr leu tonA lacY supE
thi pro hsdR- hsdM+ recA::RP4-2-Tc::Mu-Km::TnT; Stra
A(lac pro) argE (Am) rif nalA recA56 (Xpir)
thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu-Km (~.pir); Km R
recA1 Alac-pro endA1 gyrA96 thi-1 hsdR17 supE44 relA1 F' traD36
proAB + laclqZAM15
Appleyard (1954)
Simon et al. (1983)
Miller and Mekalanos (1988)
Miller and Mekalanos (1988)
Yanisch-Perron et aL (1985)
Suicide vector, contains R6K origin of replication and RP4 Mob region;
must be replicated in (;~pir) hosts; Amp R
Mobilizable vector, RSF1010 derivative, Ptac; CIm R
Mobilizable vector, pACYC184-oriT of RK2; CIm a
Origin of the km-GenBIock cassette; Amp R Km R
Plasmid vector; Amp a
cat of pACYC184 cloned into Pstl site of pJM703.1; CIm R
TnphoA carrying derivative of pJM703.1, Amp R Km R
Tn5-Tcl derivative of pJM703.1 ; Amp R Tet a
YeO3-c genomic library clone in pBR322, 8.2 kb insert; Amp R
YeO3-c genomic library clone in pBR322, t4.5 kb insert; Amp R
YeO3-c genomic library clone in pBR322, 12.4kb insert; Amp R
YeO3-c genomic library clone in pBR322, 7.3 kb insert; Amp R
The 6.4kb Hindlll-Sphl fragment of pRV3 cloned into pTM100; CIm R
The 2kb SphI-Xbal fragment of pAM100 cloned into pMMB208; CIm R
The 12kb Hindlll fragment of pRV7 cloned into pTM100; CIm R
Nrul deletion derivative of pRV16; CIm R
The 2.9 kb Clal fragment of pRV16 cloned into pRV1; CIm a
km-GenBIock derivative of pRV19, ~,trsABC::km-GenBIock; CIm R Km a
YeO3-c genomic library clone in pBR322, 7.7 kb insert; Amp R
The 5.5kb Sphl fragment of pAM100 cloned into pTM100; CIm R
Miller and Mekalanos (1988)
Morales et aL (1991)
Michiels and Cornelis (1991)
Pharmacia-LKB
Yanisch-Perron et aL (1985)
This work
Miller and Mekalanos (1988)
Cornelis et aL (1991)
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
Sequencing vectors
Sewage isolate, specific for YeO3 LPS O-antigen
Sewage isolate, isolated using YeO3-R1
The 2.3 kb Sa/I-Nrul fragment of pRV7 cloned into M13mp19
The 3.5 kb SalI-Nrul fragment of pRY7 cloned into M13mp18
Messing et aL (1981)
AI-Hendy et aL (1991b)
This work
This work
This work
Strains
Y. enterocolitica
YeO3 (= 6471/76)
YeO3-c (= 6471/76-c)
YeO3-R1
YeO3-R2
YeO3-RfbR12
YeO3-RfbR12-R
YeO3-RfbR14
YeO3-RfbR14-R
YeO3-RfbR7
YeO3-c-trs8
YeO3-c-trs8-R
YeO3-trsl 1
YeO3-trsl 1-R
Ye75S
Ye75R
E. coil
C600
$17-1
Sy327~pir
Sml 0~, pir
JM109
Plasmids
pJM703.1
)MMB208
)TM100
)UC4K
)UC18
)RV1
)RT733
)GSC62
)RV3
)RV5
)RV7
)RV11
)RV12
)RV15
)RV16
)RV17
)RV19
)RV19-GB
yAM100
)AM200
Bacteriophages
M13mp18&19
~pYeO3-12
~R1-37
t608
t609
Experimental procedures
Bacterial strains, plasmids a n d bacteriophages
Bacterial strains, plasmids and bacteriophages used in this
work are listed in Table 5, In addition, from the strain collection of the Skurnik laboratory, altogether 94 Yersinia strains
representing eight species of the genus and a number of
different serotypes were used in the phage sensitivity testing
(see Table 3). Bacteria were routinely cultured in L u r i a Bertani broth (LB) or on agar plates based on LB (LA plates).
For bacteriophage cultures tryptic soya broth (TSB) or agar
plates (TSA) were also used. For transposant selections, the
Yersinia selective agar (CIN agar, Oxoid) plates supplemented with appropriate antibiotics were used. Antibiotic
© 1995 Blackwell Science Ltd, Molecular Microbiology,17, 575-594
Bacteriophage-resistant LPS mutants of Yersinia enterocolitica 0:3
concentrations used in agar plates and broths were: ampicillin
(amp, 50pgml-1), tetracyclin (tet, 12.51~gm1-1), chloramphenicol (clm, 20 I~g ml- 1), kanamycin (km, 100 pg mlin plates and 20 pg ml-~ in broth).
Isolation of bacteriophages
Bacteriophage enrichment from raw sewage obtained from
the Turku City sewage treatment plant was carried out as
described (AI-Hendy et al., 1991b). Briefly, YeO3-Rl-reacting
bacteriophages in the sewage were enriched by adding
0.1 ml of overnight culture of YeO3-R1 (grown at 37°C) in
9 ml TSB to 3 ml sewage and incubating the mixture for 6 h.
To lyse bacteria and release phages, 3 - 5 drops of chloroform were added after which cell debris was removed by centrifugation. From this enriched phage stock, YeO3-R1 specific
phages were isolated by plating diluted stock with target bacteria in soft agar to obtain individual plaques. Well separated
plaques were replated with the target bacteria 2 - 3 times. A
concentrated phage stock was prepared by infecting 0.1 ml
of target bacteria in 9 ml TSB with the purified phage and incubating the culture until lysis occurred. After chloroform treatment and debris removal the stock was sterile-filtered through
a 0.22 pm pore size filter. One of the bacteriophages obtained
by this method was named ~R1-37. This phage formed tiny,
pinpoint, clear plaques in soft agar, in contrast to (~YeO3-12
(AI-Hendy et aL, 1991b), which formed very large plaques.
These phenomena indicated that ~R1-37 is a large and
bulky bacteriophage that would not diffuse in soft agar rapidly
and that (~YeO3-12 is a small bacteriophage that was able to
diffuse easily in soft agar.
Phage titration and specificity assays were performed by
pipetting 5-20p.I droplets of serial phage dilutions on bacterial lawns on TSA agar plates. The lawn was prepared by
flooding the plate with bacterial suspension and allowing the
agar surface to dry before adding the phage droplets. To isolate spontaneous rough derivatives of the strains, bacteriophage (~YeO3-12 was used as described (AI-Hendy et al., 1992).
591
the BLASTprograms (Altschul et al., 1990). Similar amino acid
sequences were aligned using the PILEUP program of the
GCG package, and the consensus sequences shown in the
alignments in Figs 3 - 6 were calculated by PaEn-Y using a
plurality value of (n/2) + 1 where n was the number of aligned
sequences. Thus the consensus sequence is generated
under the condition that more than half of the sequences
have similar amino acid at the same position of the alignment.
All the clonings of Yersinia DNA were carried out in the E.
coli C600 background, transformed into E. coil $17-1 and
mobilized into the Y. enterocolitica 0:3 strains. For the construction of the GenBIock-insertion mutants, the suicide plasmid pRVl derivatives were maintained in E. coli Sy327 Xpi r
background and then transformed into E. coil strain Sml0
~'pir. The plasmids were then mobilized into YeO3 and
YeO3-c, where homologous recombination events were
selected for by resistance of the colonies to kanamycin.
Double-recombinants were selected by the sensitivity of the
colonies to chloramphenicol after cycloserine enrichment
(Gripenberg-Lerche et aL, 1994; Pepe et aL, 1994).
Construction of a transposon library
Two transposon Tn5 derivatives, T n 5 - T c l (gift of G. R.
Cornelis) and Tn5-phoA (gift of V. L. Miller), were used to construct a transposon library of YeO3-c, as decribed (Cornelis et
aL, 1989). Briefly, overnight cultures of the donor (Sml0Xpir/
pGSC62 or Sm10Xpir/pRT733) and recipient (YeO3-c) were
mated overnight at 30°C using donor:recipient ratio of 5:1
on LA plates. On the next day, the bacterial lawns were collected in phosphate-buffered saline (PBS) and spread on
appropriate selective plates (CIN-km or CIN-tet). Since transposition frequency to Y. enterocofitica was low, more than 100
plates were used for selection in order to obtain a sufficient
number of independent transposants. The colonies were
pooled, the pool was amplified and stored frozen at - 7 0 ° C
in portions in 20% glycerol in TSB.
Construction of plasmids
Recombinant DNA methods
Plasmid DNA isolations, restriction enzyme digestions, DNA
ligations and transformations were performed as described
(Ausubel et aL, 1987).
Nucleotide sequencing of single- and double-stranded DNA
as template was performed using Sequenase Version 2.0
(USB), by cyclic sequencing as described (Adams and
Blakesley, 1991) or using the Circum-Vent sequencing kit
(Millipore). As templates in sequencing, pRV3, pRV7,
pAM100, t608 and t609 (Fig. 1 and Table 5) were used.
Sequence reactions were analysed either in glycerol-tolerant
sequencing gels, as recommended (Pisa-Williamson and
Fuller, 1992) or using the BaseStation automated DNA
sequencer (Millipore). Analysis and handling of the nucleotide
and amino acid sequences were performed using the GCG
Program Package (Program Manual for the GCG Package,
Version 7. April 1991. Genetics Computer Group, Wisconsin), the computer system GENEUS(Harr et aL, 1985; 1983)
and the BaseStation DNA sequence assembly manager
(Millipore). National Center for Biotechnology Information
databases were searched through the E-mail server using
© 1995 Blackwell Science Ltd, Molecular Microbiology, 17, 575-594
The genomic library of YeO3-c constructed in pBR322 was
used as a source of YeO3-c-specific clones (AI-Hendy et aL,
1991b). For trans-complementation studies and for construction of suicide plasmids, because transformation and electroporation frequency of Y. enterocolitica 0:3 is practically close
to zero, different fragments of the pBR322-based plasmids
were cloned into mobilizable vectors (see Fig. 1). Briefly, to
construct pRV12, pRV3 was digested with Hindlll and Sphl.
The 6.4 kb fragment was cloned into Hindlll/Sphl-digested
pTM100 to yield pRV12. To construct pRV15, pAM100 was
cut with Sphl and Xbal, and the 2.0kb fragment including
the trsA gene (see Fig. 1) was cloned into Sphl/Xbaldigested pMMB208. In pRV15, the trsA gene is under the
control of the inducible tac promoter. The upstream part of
the 2 kb fragment contained about 200 bp of pBR322-derived
sequence (from between the Sphl and BamHI sites of
pBR322), pRV16 was constructed by digesting pRV7 with
Hindlll and cloning the 12kb fragment into Hindlll-digested
pTM100. Also, the 12 kb fragment included about 300 bp of
pBR322-derived sequences (from between the Hindlll and
BamHI sites). The 12kb Hindlll fragment of pRV7 was
592
M. Skurnik, R. Venho, P. Toivanen and A. AI-Hendy
Table 6. Tn5 and pACYC184-specific oligonucleotidesUsed in this work.
Oligonucleotide
5'-3' nucleotide sequence
5' position
3' position
Use
Accession number/
target
Tn5-V
Tn5-S
Tn5-P
Tn5-O
MS-27
MS-28
gacgctacttgtgt at aagagt cagg
gagcagaagtt at catgaacgtta
62
144
660
65
396
3652
38
121
636
89
375
3671
Ligation-PCR
Sequencing
PCR
Ligation-PCR
PCR
PCR
V00615/Tn5
V00615/Tn5
V00615/Tn5
V00615/Tn5
X06403/pACYC184
X06403/pACYC184
tgcccatgcgt aaccggct agttgc
gaacggaaccttt c c c g t t t t ccag
t cgctgcagaat aaat aaat cctggtg
atgctgcagcaat agacataagcggc
cloned into pTM100 in such a way that the Nrul site at 10.4 kb
(Fig. 2) was oriented towards the single Nrul site of pTM100
located in the tet gene. pRV17 was derived from pRV16 by
Nrul digestion and religation so that the 4 kb Nrul fragment
was deleted, pAM200 was constructed by cloning the 5.5 kb
Sphl fragment of pAM100 into Sphl-digested pTM100. The
same pBR322-derived 200bp sequence as in pRV15 was
included in pAM200. Both t608 and t609 were constructed
by cloning the purified 2.3 and 3.5 kb SalI-Nrul fragments
of pRV7 into M13mp19 and M13mp18, respectively; t608
and t609 were maintained in E. cofiJMl09.
pRV1 is a CIm R derivative of the suicide vector pJMT03.1
and it was used to construct genomic insertion derivatives
by marker exchange. To construct pRVl, the cat gene of
pACYC184 was amplified by PCR using primers MS-27 and
MS-28, which generated Pstl sites at the ends of the amplified fragment (Table 6). The fragment was digested with
Pstl and cloned into the single Pstl site of pJM703.1, pRVl
can be used as a suicide vector and it contains single sites
for EcoRV, Sphl, Sail and Clal, and no sites for Nsil. To construct pRV19, the 2.9kb Clal fragment of pRV16 (between
positions 2.7 and 5.6 kb, Fig. 2) was cloned into the single
Clal site of pRVI. pRV19 was digested with Nsil, which
released two small Nsil fragments from inside the 2.9kb
Clal fragment, and a Pstl-digested Kin-resistance GenBIock
was cloned between the Nsil sites to obtain pRV19-GB.
PCR and ligation-PCR
Standard PCR amplification was performed as described
earlier (Saiki et al., 1988), in 0.5 ml polypropylene tubes with
a Perkin-Elmer Cetus thermal cycler. The PCR conditions
were as follows: 100 ~1 of reaction mixture contained template
and primers (10-100pmol per reaction), 2.5 units Taq polymerase (Perkin Elmer Cetus), 200 I~M of each dNTP, 50 mM
KCI, 10mM Tris-HCI, pH 8.3, 1.5mM MgCI2 and 0.1 mgml - I
gelatin. The reaction mixture was then overlaid with mineral
oil. The PCR steps (94°C for 45 s, 63°C for 1 min, 72°C for
2 min) were repeated 22-30 times as specified. The primers
are listed in Table 6.
A modified ligation-PCR method (Rich and Willis, 1990)
was used. Two primers located tail-to-tail in the inverted
repeat of Tn5 were designed (Tn5-O and Tn5-V, Table 6).
For the ligation-PCR, genomic DNA isolated from YeO3RfbR12 was completely digested with Rsal or Hpall. These
restriction enzymes have recognition sites a few hundred
base pairs inside the IS50 sequence of Tn5-Tcl. The generated restriction fragments were ligated in a very diluted
reaction (1 ng DNA in 200 I~1).A 0.25 ng sample of the ligated
material was used as a template in PCR, which contained 30
cycles. Two amplified fragments were obtained from both ligations representing both ends of the transposon and their flanking YeO3-RfbR12 DNA. For the Rsal-digested template, the
two fragments were both about 400bp in size, and for Hpall,
1.2 and 1.4 kb (not shown).
Antibodies
Mouse monoclonal antibodies (Moab) 2B5, specific for the
YeO3 LPS core, and A6, specific for the YeO3 LPS Oantigen, have been described earlier (AI-Hendy et aL,
1991a,b; Pekkola-Heino et aL, 1987).
Isolation and analysis of LPS
LPS isolations by the hot phenol-water procedure were
performed from bacteria grown overnight at 22°C in LB
medium. The small-scale procedure is described in Zhang
and Skurnik (1994), and the large-scale procedure, in Helander (1985). LPS was analysed in SDS-PAGE as described
(Zhang and Skurnik, 1994) or in deoxycholate (DOC)PAGE as described (Krauss et aL, 1988). Silver staining of
the gels was performed as described (Zhang and Skurnik,
1994). For use as controls in DOC-PAGE analysis, purified
LPS from Salmonella minnesota R60 (Ra chemotype
mutant), R5 (Rc chemotype mutant), R595 (Re chemotype
mutant), Y. enterocolitica Ye75S, and Ye75R were kindly
donated by Dr Hubert Mayer (Freiburg, Germany), who had
originally received the Salmonella LPSs from Dr Otto L(~deritz
(Freiburg, Germany).
Acknowledgements
This work was supported by the grants from the Academy of
Finland, the Turku University Foundation and the Sigrid
Jus~lius Foundation. Joanna Radziejewska-Lebrecht and
Hubert Mayer are thanked for the generous gift of LPS
samples, and Virginia Miller and Guy Cornelis for plasmid constructs. Renato Morona, Joseph Lam and Joanna Goldberg
are thanked for allowing us to refer to results prior to
publication.
Note added in proof
Based on more recent analysis (J. Radziejewska-Lebrecht, M.
Skurnik, A. S. Shashkov, and H. Mayer, unpublished), the
schematic drawing in Fig. 7 on Y. enterocoh'tica 0 : 3 LPS
© 1995 BlackwellScience Ltd, MolecularMicrobiology,17, 575-594
Bacteriophage-resistant LPS mutants of Yersinia enterocolitica 0"3
contains two major errors. (i) In the outer core, Sugar 4 should
be linked to Sugar 2. (ii) In the inner core, a second KDO residue is linked to the first KDO residue. In addition, one of the
residues of the outer core is N-acetylfucosamine, which is significant for the discussion of the roles of TrsF and TrsG (see
the Discussion).
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