Molecular Microbiology (1991) 5(3), 585-593
ADONIS 0950382X9100066K
Intervening sequences (IVSs) in the 23S ribosomai RNA
genes of pathogenic Yersinia enterocolitica strains. The
IVSs in K enterocolitica and Salmonella typhimurium
have a common origin
M. Skumik* and P. Toivanen
Department of Medical t/icrobiology, Turku University,
SF-20520 Turku, Finland.
Summary
The 23S ribosomal RNA (rRNA) was shown to be in two
fragments in pathogenic Yersinia enterocolitica. The
cleavage site in the structural gene of the 23S rRNA
was occupied by an intervening sequence (IVS) of
about 100 nucleotides, analogous to IVSs found
in salmonellae (Burgin ef al., 1990). Nucleotide
sequences of IVSs of several Y. enterocolitica strains
revealed that the IVSs of the highty virulent Y. enterocolitica serotypes strains, and the IVS of Salmonella
typhimurium were about 90% similar. On the other
hand, the IVSs of the highly and the poorty virulent Y.
enterocoiitica serotypes were only about 60% similar.
These results give the impression that at some point
during the IVS evolution, the highly virulent Y. enterocolitica and S. typhimurium both received their IVSs at
about the same time from the same source, and that
the fworty virulent serotypes received their IVSs earlier. We also found that strain LB5010, derived by
extended mutagenization of S. typhimurium LT2, had
lost the IVSs originally present in LT2, and that this loss
had created a new 'hairpin loop' which substituted for
the original 'hairpin loop'.
Introduction
When performing Northern blot analysis with different
Yersinia strains, the lack of intact 23S ribosomai RNA
(rRNA) in two Yersinia enterocolitica strains, of serotypes
0:3 and 0:8. was observed, tn addition to intact 16S rRNA
of 1550 nucleotides, and instead of the 23S rRNA, two
smaller rRNA species of approximately 1750 and 1150
nucleotides in size were seen in these V. enterocolitica
strains. The sizes suggested that they were cleavage
products of 23S rRNA. This fragmentation and the sizes of
Received 11 September. 1990; revised 5 November, 1990. "For
spondence. Tel. (21) 6337407; Fax (21) 330008.
the fragments resembled that reported in Salmonella
typhimurium (see below). In contrast, Yersinia pestis and
Yersinia pseudotuberculosis strains had a normal nontragmented rRNA pattern similar to that of Escherichia coli
{pattem A).
In general, rRNA In prokaryotes consists of three
species: 23S, 16S, and 5S rRNA (King et ai, 1986). The
genes of these molecules, which contain approximately
2900, 1550 and 120 nucleotides, respectively, make up
operons (Noller and Nomura, 1987). In prokaryotes there
are 1 -10 such ribosomai DNA (rDNA) operons, the number
correlating to the growth rate; in Escherichia coli and other
enterobacteria there are seven (King et ai. 1986). The
operons are transcribed as one transcript which is then
processed into the three rRNA species (Noller and
Nomura, 1987). The processing is mainly due to RNase III.
with some participation of a 3'-exonuclease (King et ai,
1986).
With a few exceptions, the prokaryotic 23S rRNA
molecules have been found to be similar to the E. coli
counterpart. The exceptions are the 23S rRNA of
Rhodopseudomonas spheroides.
Rhodopseudomonas capsulata (reviewed by Pace, 1973), of Leptospira
interrogans (Hsu et ai. 1990), and of many salmonellae
(Winkler, 1979: Smith et ai. 1988; Burgin et ai. 1990),
which have been found to be fragmented. In S. typhimurium and Salmonella arizonae. the 23S rRNA fragmentation was shown to be due to the presence in the
rDNA of extra sequences about 80-90 nucleotides
long (Burgin e^ ai, 1990), which the authors called
intervening sequences (IVSs), although they are not
true introns. In S. typhimurium the IVS was located
1170 nucleotides from the 5' end of the 23S rRNA
molecule, whereas S. arizonae had two IVSs, one
being similar to the S. typhimurium IVS at position
1170, and the other being non-identical at position 550.
Fragmentation resulted from the action of RNase III on
the stem-loop structure formed by the IVS in the
transcribed 23S rRNA. On the other hand, the 23S
rDNA of the archaebacterium
Desulfurococcus
mobilis was shown to contain a true intron, which is
spliced off from the 23S rRNA (Kjems and Garrett.
1985).
586
M. Skurnik and P. Toivanen
23SrRNfl
l6SrRNA
In this report, we demonstrate that Y. enterocolitica
rDNA contains IVSs analogous and highly similar to the
IVSs of Salmoneiia at position 1170, indicating a common
evolutionary origin for the IVSs of these two enteric groups
of pathogens. We also demonstrate that IVSs are found in
all pathogenic Y. enterocolitica strains and in two Yersinia
bercovieri 0:58,16 strains, but not in the so-called
environmental Y. enterocolitica strains, and, as a rule, not
in the other Yersinia speoies.
Results and Discussion
Distribution of strains with fragmented 23S rRNA among
Yersinia species
Y. enterocolitica is a heterogenous collection of highly
virulent (lethal to mice), poorly virulent (non-lethal to mice),
or avirulent (=environmental) strains, which belong to
several biotypes and serotypes. The highly virulent strains
are isolated mainly in the USA, whereas the poorly virulent
strains are generally encountered in other parts of the
world. The distribution of the fragmented 23S rRNA
pattern (pattern B) was studied among the Y. enterocoiitica strains of different biotypes and serotypes, as well as
among many other Yersinia species.
To this end, RNA was isolated from 47 Yersinia strains
and from seven other bacterial species including Salmonella strains for comparison (Table 1), and the rRNA
patterns were analysed in gel electrophoresis (Fig. 1).
Pattern B was only seen in the strains belonging to the
pathogenic serotypes of Y. enterocolitica. Mixed pattern
AB was, however, seen in all six Y. enterocolitica 0:8
strains, and in two Y. bercower/0:58,16 strains (3016/84
and 3984/84, lanes 4 and 12. Fig. 1), and in two out of four
Fig. 1. Agarose gel electrophoretic patterns of
bacterial rRNA. The locations of the 23S and 16S
rRNA bands are indicated on the right. Bacterial
stains were (see also Table 1): lane 1, Ye {Y.
enterocolitica) C36; lane 2, Ye 3229; lane 3, E.
coli LE 392; lane 4, K t>ercovierl 3016/84: lane 5.
Shigella flexneri\ lane 6. Ye aO81-c; lane 7. Ye
8081; lane 8. Ye 6471/76-c; lane 9, Ye 6471/76;
lane 10. Ye 2139/72; lane 11, Ye E736; lane 12,
Y. bercoWen 3984/84; lane 13, Ye 671/80; lane
14, K pset>doftj£)e/cutos/s YPIII/plBI.
Salmonella strains studied (Table 1). incidentally, the
fragmentation of the 23S rRNA of S. typhimurium and K
enterocoiitica 0:8 strains was similar (data not shown).
A likely explanation for the observed fragmentation is
that the pattern B strains contain at the cleavage site a
specific sequence which can be attacked by RNase III, as
was shown to happen with S. typhimurium (Burgin et ai,
1990). This is supported by the finding of strains with
mixed pattern. Since eubacteria possess several rDNA
operons (King etai, 1986), and in Y. enterocolitica. as in
other Enterobacteriaceae, there are seven rDNA operons,
the mixed pattern would be due to the heterogeneity of the
operons in these strains. Some of the operons produce
pattern A non-cleaved 23S rRNA, and others the pattern B
cleavable 23S rRNA. The sequencing data verified that the
mixed pattern AB is real (see below).
No association of pattern B of 23S rRNA with the
virulence piasmid ofi. enterocolitica
As pathogenic Yersinia harbour a 65-70 kb virulence
ptasmid (reviewed by Perry and Brubaker, 1983; Skurnik,
1985; Cornelis e/a/., 1989) it could be possible that pattern
B and the cleavage activity were associated with the
plasmid. This was not the case, since plasmid-cured
isogenic strains also had pattern B (Fig. 1, lanes 6-9; Table
1)Location of the cleavage site
To find the location of the cleavage site in the 23S rRNA,
oligonucleotides MS-2 and MS-3 were used as probes in
Northern blotting experiments with pattern A, B, and AB
strains. The probes were chosen so that hybridization of
23S rRNA cleavage in Yersinia and Salmonella
587
Table 1. Ribosomai RNA patterns of various bacterial strains.
Bactefium
Strain
0-serotype
Virulence
plasmid
rRNA
pattern'
IVS group""
Source and/or Reference
Y. pestis
EV76
Y. pseudotuberculosis 2812/79
YPIII(plB1)
324/80
1
III
111
A
A
A
A
Lab. strain
IV
A
Lab. strain
V
A
Y. intermedia
821/84
9/S5
52.54
16,21
A
A
Y. fred&iksenii
3400/83
38/83
119/84
404/81
OMBL3
16
48
12,25
16
A
A
A
A
A
Portnoy and Falkow (1981)
Skurnik (1984)
Bolin et al. (1982); Gemski ef at. (1980)
Human isolate, Dept. Medical Microbioiogy,
University of Oulu, Finland
Laboratory reference strain. Public Health Lab.
Helsinki. Finland
Laboratory reference strain. Public Health Lab.
Helsinki. Finland
Skgmik(1985)
Human isolate, Dept. Medical Microbiology,
University of Oulu. Finland
Skurnik (19S5)
Skurnik (1985)
Skumik (1985)
Skumik (1985)
Fish isolate. Dept. Medical Microbiology,
92/84
127/84
3016/84
59(20,36,7)
NT
58.16
A
A
AB
University of Oulu, Finland
Skurnik (1985); Wauters et al. (1988)
Skumik (1985); Wauters et al. (1988)
Human isolate. Dept. Medical Microbiology.
Y. kristensenii
Y. ruckerii
Y. mollaretii
Y. bercoviari
V. antsnxxlifica
continued overleaf.
3984/84
58,16
132
1
1142
2
6471/76
6471/76-c
E701
7500
671/80
22227/80
155/84
6,30
6,30
6,31
1309/80
605/80
8081
8081-c
NY81-66
WA
8339
FRI-Vel
Ruokola/71
3102/80
5081
9312/78
036
874/77
E736
431/64
6,31
7.8
8
8
8
8
8
8
9
10
13a, 13b
13.18
15
20
21
25
A
A
AB
AB
AB
AB
AB
AB
B
A
B
B
B
B
B
5186/84
2139/72
248/84
626/63
3229
26.50
34
35.52
41(27),42
50
A
2a
AB
2a
3
B
3
B
B
B
1
1
1
1
4,32
5,27
A
A
A
2a
2a
2a
2a
2a
2a
1
2b
2b
1
2b
2b
A
B
A
A
A
2b
University of Oulu, Finland; Skumik (1985);
Wauters dfaA (1988)
Human isolate. Dept. Medical Microbiology,
University of Oulu. Finland, Wauters ef al.
(1988)
H. H. Moilaret, Institut Pasteur, Paris, France,
through G. Kapperud, Oslo. Norway
H. H. Mollarel, Institut Pasteur, Paris, France,
through G. Kapperud, Oslo, Norway;
Kapperud ef a/. (1985)
Skumik (1984)
Skurnik (1984)
Pen7 and Brubaker (1983)
H. H. Mollaret, Institut Pasteur, Paris, France.
through G. Kapperud, Oslo. Norway;
Kapperud df a/. (1985)
Skumik (1984); Skumik (1985)
Skurnik (1964); Skurnik (1985)
Human isolates. Dept. Medical Microbiology,
University of Oulu, Finland; Skumik (1965)
Skumik (1984); Skumik (1985)
Skumik (1984); Skumik (1985)
Portnoy ef a/. (1981)
Portnoy efa/, (1981)
Kapperud etal- (1965)
Carter (1975)
Toma efa/. (1984)
Doyle efa/. (1981)
Skumik (1964)
Skurnik (1984)
Toma efa/. (1964)
Kay efa/. (1983)
Perry and Brubaker (1983)
Kayetaf. (1983)
Perry and Brubakar (1983)
Human isolate. Dept. Medical Microbiology.
University Oulu, Finland
Skumik (1985)
Kay efa/. (1983)
Skumik (1985)
Skumik (1985)
Skumik (1985)
588
M. Skurnik and P. Toivanen
Table 1. (continued).
Bacterium
Strain
E. coll
Salmonetta
LE392
Salmoneita
typhimurium
LB5010
Satrrjonella typhi
0-serotype
Virulence
plasmid
rRNA
pattem^
IVS group"
A
AB
ATCC 13311
3851
AB
A
Shigelta flexneri
Vibrio choterae
Source and/or Reference
Kushner (1967)
Human isolate. Public Health Lab., Turku,
Finland
Received from P. Helena Makela, Helsinki,
Finland
2a
Human isolate. Dept. Medical Microbiology,
Turku University, Turku, Finland
Ouality control strain, Central Public Health
Laboratory, Division of Microbiological
Reagents S Ouality Control, London, UK
Ouality control strain. Central Public Health
Laboratory, Division of Microbiological
Reagents & Quality Control. London. UK
a. rRNA pattems: A = 23S + 16S rRNA; B = 16S + fragmented 23S rRNA; AB = 23S + 16S + fragmented 23S rRNA.
b. iVS groups; 1 = IVS of YeO3-type present in all rDNA operons; 2a - IVS of S. typhimurium-type present in some rDNA operons; 2b - IVS of S.
typhimuriurrhtype present in all rDNA operons.
the probes to different 23S rRNA fragments would indicate
that the cleavage site is located 1750 nucleotides from the
5' end of the 23S rRNA molecule, whereas hybridization of
both probes to the 1750 nucleotide fragment would
indicate that the cleavage site is located 1150 nucleotides
from the 5' end. Both oligonucleotides hybridized to the
1750 nucleotide fragment (Fig. 2A), demonstrating that the
cleavage site was located 1150 nucleotides from the 5'
end of the 23S rRNA.
In order to locate the cleavage site exactly, the oligonucleotide MS-2 was subsequently used as a primer in RNA
sequencing reactions {Fig. 3). The sequencing reaction
stop in Y. enterocolitica 6471/76 (hereafter referred to as
YeO3) showed that the cleavage site was located between
nucleotides 1171 and 1172 of the £ coli 23S rRNA. This
coincided exactly with the location of the cleavage site
found in Salmonella (Burgin etai. 1990).
Detailed analysis of the cieavage region
The 23S rRNA of Y. pestis EV76, Y. pseudotuberculosis
YPIII/plBI, and Y. enterocolitica 671/80 were sequenced
by RNA sequencing over the cleavage region using MS-2
23S16S-
as primer (Fig. 4). For pattern B strains, DNA sequencing
over the corresponding region of the gene was used. This
was initially performed using the genomic DNA of YeO3 as
template, and later using polymerase chain reaction
(PCR)-amplified DNA as template (see below). With direct
genomic sequencing, a 60-70-nucleotide sequence of
YeO3 was obtained, which revealed that the sequence of
YeO3 diverged from that of E co//about ID nucleotides
before the cleavage site and that the sequence after the
cleavage site had no similarity to the whole E. coli 23S
rRNA sequence (Fig. 4).
However it was not obvious whether the genomic
YeO3-specific sequence was present in the smaller 23S
rRNA fragment, i.e. was the rRNA cut between nucleotides
1171 and 1172, or had part of the sequence disappeared
during cleavage, and it was unclear whether the sequence
upstream of the cleavage site in pattern A strains is found
elsewhere in the 23S rRNA of the pattern B strains. To this
end, three oligonucleotides, MS-6, MS-7, and MS-8, the
latter being complementary to the YeO3-specific
sequence, were used as probes in Northern blottings. The
E. co//-derived probes, MS-6 and MS-7, gave the same
hybridization pattern; that of MS-6 is shown in Fig. 2 (panel
Fig. 2. Northern blotting of the RNA samples with
23S rRNA specific oligonucleotide probes.
Bacterial strains were: lane 1, E. coti LE 392; lane
2, V. pBstis EV76; lane 3, Y. pseudotuberculosis
YPIII/plBI; lane 4, Y. enterocolitica 67^/B0\ lane
5. V. enterocolitica 6471/76; lane 6, Y. bercovieri
3016/84. Hybridization with the oligonucleotide
probe MS-2 (MS-3 gave an identical pattem) is
shown in panel A, and with MS-6 (MS-7 gave an
identical pattern) in panel B. Hybridizations for
panels A and B were performed on same
nitrocellulose filter, and the panels are mirror
images of each other. The positions of 23S and
16S rRNA are indicated between the panels.
23S rRNA cleavage in Yersinia and Salmonella
589
probably have no role in the structure and function of the
50S ribosomai subunit. Since protein-protein interactions
have been shown to play an important role in the cohesion
of the 50S ribosomai subunit (Roth and Nierhaus, 1975),
cleavage of the 23S rRNA backbone at certain sites might
not affect its functional properties.
Overexposure of Northern hybridizations, like those
in Fig. 2, revealed that a minute fraction of the 23S rRNA
extracted from pattern B strains was intact. This fraction
must represent the newly synthesized and still uncleaved
23S rRNA. However, the stage when the protnjding IVS
loop is degraded remains to be elucidated.
flCGT
1190 —
1200 -
f
1210-
1220 —
1230
Fig. 3. RNA sequencing of (1) £ ccrii LE 392 and (2) YeO3.
Oligonucleotide Ms-2 was used as primer. The numbering on the left
refers to the nucleotide positions in mature 23S rRNA of E. coli. position
1172beingmarkedvnth a dot (Noller and Nomura. 1987). In the figure.
for the sake of the E. coli sequence, the end of YeO3 sequence is
overexposed. Accurate mapping of the cleavage site was performed
using less-exposed autoradiograms.
B). Both also hybridized to the smaller fragment of pattern
B and of the mixed pattern strains (Fig. 2B, lanes 5 and 6).
MS-8 did not, however, hybridize to any of the rRNA bands
(not shown). This suggested that the rDNA genes of the
pattem B strains contain IVSs analogous to salmonellae
(Burgin etai, 1990), which are not present in mature 23S
rRNA.
To verify that the oligonucleotide MS-8 sequence was
indeed present in the genomic DNA of pattern B strains,
the genomic DNA from YeO3 was digested with SamHI
and EcoRI and analysed by Southern blotting using
oligonucleotides MS-6 and MS-8 as probes. Both probes
hybridized to bands of the same size in both digestions,
respectively, suggesting that both sequences are closely
located in the chromosome. Southern analysis of pattern
A genomic DNA, on the other hand, revealed only the
presence of MS-6-speciflc sequences (data not shown).
The results achieved pointed to the possibility that the
extra nucleotides of the IVS in the pattern B 23S rRNA
were located in the assembled 50S ribosomai subunit as a
loop projecting out of the surface. This loop would be
easily accessible to cellular RNases. The loop would
Nucleotide sequences of the IVSs
Genomic DNA was amplified with PCR using oligonucleotides MS-2 and MS-11. or MS-2 and MS-13, as primers.
These primers amplified 712 and 158bp fragments,
respectively, from E coli. and a fragment about lOObp
longer from pattern B strains (data not shown). This size
was close to the size of IVSs found in salmonellae (Burgin
etai. 1990), indicating, together with the identical location
of the IVS, that Y. enterocolitica and salmonellae posses
analogous IVSs.
The nucleotide sequences of the IVSs were determined
using the shorter PCR-amplified fragments as templates,
and labelled MS-2 as primer. Since there are seven rDNA
operons in Enterobacteriaceae, the PCR-amplified products represented all these. The sequencing gels of
pattern A and B strains were specific, implying that all the
operons are highly similar at the sequenced region. In the
sequencing gels of the pattern AB strains the situation
was, of course, different. The reading of the IVS
sequences was. in most cases, possible, since usually the
sequence stops of the IVS-containing operons (IVS-operons) dominated over those of the non-IVS opercns. This
indicated that most of the operons contained IVSs. With
the primers used, the accumulation of sequencing stops
at the end of the 158-nucleotides non-IVS-derived
sequences, however, prevented the reading of about 10
nucleotides of the IVS sequence. This was circumvented
by running a second PCR reaction using as templates,
separately, the two bands of the first PCR amplification.
Agarose gel slices containing the bands were crushed into
200 \L\ of aqua, and 10|xl of the cleared supernatant was
used as template for the second PCR reaction. This
approach allowed the sequencing of both the IVS and
non-IVS operons of a single strain.
The IVS sequences are shown in Fig. 4. Comparison of
the IVSs revealed two groups of V. enterocolitica strains
within which the IVSs are closely related. Group 1 is
formed by the poorly virulent Y. enterocolitica strains
including serotypes 0:1, 0:2. 0:3. 0:5,27, 0:9 and 0:15.
The IVSs within group 1 were 99bp long, and >99%
590
M. Skumik and P. Toivanen
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identical (comparisons of IVSs were performed between
the nucleotides forming the complete stem-loop structures, i.e. included were nucleotides between positions
1164 and 1185 of the £ coli numbering in Fig. 4). To group
2 belong the highly virulent strains including serotypes
0:4,32. 0:8. O:13a,13b. 0:13.18. 0:20, 0:21 and 0:34.
The IVSs within group 2 were 87 bp long, and >99%
identical. The IVSs between groups 1 and 2 were about
60% identical. Group 2 strains were further divided into
two subgroups depending on the number of rDNA operons containing the IVS. To group 2a belong those strains,
such as Y. enterocolitica 0:8, which have one or more
non-IVS operons. and to group 2b those strains which
have only IVS operons (Table 1). Interestingly, the IVS of Y.
bercovien. which was present apparently in only one rDNA
operon. was almost identical to group 2 IVS. Y. t>ercovieri
is considered as non-pathogenic, and it lacks the
virulence plasmid. The two 0:58,16 strains were isolated
from human stool samples, but no disease association
was reported (Skumik. 1985). In any case, the fact that the
IVS is identical to that of the highly virulent V. enterocolitica suggests that the IVSs of these strains have a
common origin.
Oomparison of the IVSs of Yersinia with those of S.
typhimurium and S. arizonae (Burgin et ai, 1990) revealed
that group 2 IVSs were very close to the IVSs of S.
typhimurium (89% identical) and S. arizonae (84%). On the
other hand. IVSs of the two salmonellae were 85%
identical. Group 1 IVSs were 61 and 66% identical to S.
typhimurium and S. arizonae IVSs, respectively. These
results are very striking since they imply that the IVSs of
group 2 Y. enterocolitica are closer to Salmonelia IVSs
than to the IVSs of group 1 Y. enterocolitica. This is not
consistent with the overall chromosomal DNA-homology
data. DNA homology between different Y. enterocolitica
strains is >60%. whereas that between Y. enterocolitica
and salmonellae is about 20% (Bercovier et ai. 1980),
indicating that the IVS has been introduced by honzontal
transmission into these bacteria late in evolution.
The Salmonella IVSs. No IVSs in S. typhimurium LB5010
We also wanted to confirm our results by sequencing IVSs
of salmonellae. To that end we chose two well-known S.
typhimurium strains. ATCC13311 and LB5010 (Table 1). A
surprising finding was the absence of IVSs in S. typhimurium LB5010. while the ATCC13311 strain contained an
IVS 100% identical to that of S. typhimurium ATCC23566
(Burgin ef ai. 1990). LB5010 is a laboratory strain derived
from wild-type strain LT2 and it contains several mutations
{its genotype is trpC2, metA22. Hi-b. H2-e,n,x nml'
{Fels2) . fla-66, rpsL 120, xyl-404, metE551, hsdL6, hsdSA29, hsdSB121. ilv-452. leu-3121. galE856). which
have been achieved by exposing the strain to a number of
591
treatments with ethyl methanesulphonate and nitrosoguanidine as well as to several different selection procedures (Luria and Sluit. 1987). Since the wild-type strain
LT2 has an IVS (Winkler, 1979; Smith et ai. 1988; Burgin ef
ai, 1990), it can be assumed that some or all of these
treatments have induced the 'curing' of the IVSs from the
rDNA operons. It would be interesting to determine which
of the selection procedures could induce loss of IVSs from
the wild-type strain.
The simplest explanation for tVS curing would be that
during mutagenesis the activated recombination machinery has replaced the IVS operons with a non-IVS operon.
This would mean that the sequence of the LB5010 at the
cleavage region should be identical to that of S. typhimurium non-IVS operons. Comparison of the sequences
showed, however, that the LB5010 sequence at the
cleavage region is unique (Fig. 4). The 1164-1185 'hairpin'
of E. coli was replaced by a completely new hairpin in
LB5010. which was. in addition, three nucleotides shorter.
Furthermore, the LB5010 sequence contained a one-base
insertion at position 1131 (Fig. 4). Sequencing of the
non-IVS operon of ATCC13311 showed that it diverged a
little from that of E. coli. Very surprising, however, was the
observation that the IVS and non-IVS operons of a single
strain, ATCC13311. were significantly different between
positions 1211 and 1222 (Fig. 4).
Secondary structures formed by the IVSs
The Yersinia IVSs formed hairpin structures analogous to
those of the Salmonella counterpart (not shown). It was
remarkable that both ends of the IVSs forming the
14-nucieotide stem of the hairpin structure were 100%
conserved in all IVSs studied, indicating that the stem
sequence is crucial for the correct excision by RNase III
(Burgin ef ai. 1990), and may also have functional
relevance.
The primary sequence of the non-IVS operons revealed,
on the other hand, that the 1164-1185 hairpin loops in
general vary quite a lot, indicating that this loop does not
carry out any sequence-related function in the ribosomal
large subunit (LSU).
Yersinia-spec/Wc considerations
The results presented in Table 1 indicate that all the
pathogenic strains of Y. enterocolitica contain IVSs. but
the non-pathogenic strains do not. This, together with the
knowledge that the pathogenic and non-pathogenic Y.
enterocolitica differ, in addition to the many virulenceassociated properties in many other phenotypical
respects, brings up the taxonomical question of placing
the non-pathogenic strains of Y. enterocolitica outside Y.
592
M. Skumik and P. Toivarwn
enterocolitica sensu stricto. However, the overall DNAhomology data between the pathogens and nonpathogens do not support the division (Bercovier ef ai,
1980).
In the mature 23S rRNA of both pattern A and B Yersinia
strains there are Vers/n/a-specific nucleotides at positions
(E CO//numbering) 1209-11,1219-20. and 1229-30 (Fig.
4). Analysis of the changes reveals that they are in the E.
coli hairpin formed by nucleotides 1198-1247 (Noller and
Nomura. 1987). Nucleotides 1209-11 are part of a bulge,
and nucleotides 1219-20 and 1229-1230 complement
each other, in the hairpin stem.
Our results also suggest that the Y. enterocolitica
0-side-chain specificities are evolutionally younger than
the appearance of IVSs, since within groups 1 and 2 the
IVSs are >99% identical. It is very unlikely that the IVS has
been introduced into several stains representing different
serotypes at the same time. It is more likely that different
serotypes have evolved after the IVS has settled into the
23S rDNA gene.
Biological relevance and evolutionary implications
A mysterious question is the biological role of IVS. if there
is one. Can it be a coincidence that IVSs are found
conserved in these two groups of enteric pathogens? If
not, what kind of benefit could IVSs have for these
bactena? One hypothetical but intriguing role could be
resistance to bacteriocins elaborated by the normal flora
of the gut (Mason and Richardson, 1981). It is known, for
example, that bacteriocins colicin E3 and colicin DF13
attack and cleave 16S rRNA. thus inhibiting protein
synthesis (reviewed by Luria and Sluit, 1987). One could
envisage that some unknown bacteriocin uses the native
23S rRNA loop at position 1164-1185 as target. If the
bacteriocin itself is not an RNase, it could function by
rendering the loop at the cleavage site accessible to
cellular RNases. Altering the target by IVSs would make
the loop stem, after the clean removal of the IVS by RNase
III, resistant to any RNAse activity.
The results of this work, even if the role of the IVS
remains to be elucidated, provided evidence that in
nature, between the members of the two enteric genera,
there exists horizontal exchange of genetic material,
possibly via plasmids or bacteriophages.
Isolation and analysis of ribosomal RNA
RNA was isolated by the hot-phenol extraction method (Scherrer
and Damell. 1962) from 10ml of bacterial curtures. Three micrograms of the RNA samples were run in the presence of formaldehyde in 1.2% agarose gels and stained with ethidium bromide
(Ausubel efa/.. 1967).
DNA techniques
Agarose gel electrophoresis of ONA and RNA, Northem and
Southem blottings, restriction digestions, ligations. transformations, and preparation of genomic DNA were performed
using standard methods (Ausubel efa/., 1987). Hybridization with
oligonucleotide probes was performed as described (Albretsen ef
al., 1988), with a formamide concentration of 15% in the
hybridization mix.
Oligonucleotides
The following oligonucleotides were used in this work as primers
and probes (in parentheses are the nucieotide positions in E. coli
23S rRNA; Noller and Nomura, 1987): MS-2, 5-ttact tatgt cagca
ttcgc acttc tg-3' (1269-1243); MS-3, 5'-gcaca gtgct gtgtt tttaa
taaac agttg-3' (1800-1771); MS-6, 5'-cgtcg ctgcc gcagc ttcgg
tgcat ggttt agccc c-3' (1171-1136); MS-7, 5'-catcc accgt gtacg
cttag tcgct taacc-3' (30-1); MS-8.5-ttaaa acgcctgttc gtttg tcagc
acacc ggcc-3' (complementary to 5'-end of YeO3 IVS, Fig. 4);
MS-11. 5'-ctgcg tacct tttgt ataat gggtc agcga-3' (557-586);
MS-13. 5'-gtcgg cctgc gcgga agatg t-3' (1112-1133).
Ribosomal RNA sequencing
The sequences of V. pestis EV76, Y. pseudotutierculosis YPIII/
pIBI, and Y. enterocolitica 671/80 (serotype 0:6.30) were
determined by RNA sequencing (Lane et at.. 1985). Oligonucteotide MS-2 was used as primer.
Genomic DNA sequencing
The sequence of the cleavage site of YeO3 was determined by
plasmid sequencing (Chen and Seeburg. 1985) with MS-2 as
primer and 10 M:g of genomic DNA as template.
Polymerase chain reaction and sequencing
PCR and sequencing of the PCR-amplified DNA were perfomried
using the modification described previously (Meltzer ef ai. 1990).
Oligonucleotides MS-2 and MS-13 (and in some PCR experiments, MS-11) were used as primers. The PCR-amplified DNA
fragments were analysed in 1.5% agarose gels, and stained with
ethidium bromide.
Experimental procedures
Bacterial strains
Computer programs
Bacterial strains used in this work are listed in Table 1. For RNA
isolations bacteria were grown ovemight in 10mt of Luria broth at
rown temperature.
The nucieotide sequences were handled and analysed by using
the Sequence anatysis software package of the Genetics
Computer Group, Version 6.1 (Devereux etai. 1984).
23S rRNA cleavage in Yersinia and Salmonella
Acknowledgements
We thank David J. Lane, Gene-Trak Systems, Framingham,
Massachusetts, for critical review of the manuscript, and Ms Reija
Venho for excellent technical assistance. This work was supported by the Emil Aaltonen Foundation, and the Sigrid Jus6lius
Foundation.
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