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Journal of Bacteriology, May 2001, p. 2852-2858, Vol. 183, No. 9
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.9.2852-2858.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Low-Molecular-Weight Plasmid of Salmonella
enterica Serovar Enteritidis Codes for Retron Reverse
Transcriptase and Influences Phage Resistance
I.
Rychlik,1,*
A.
Sebkova,1
D.
Gregorova,1 and
R.
Karpiskova2
Veterinary Research Institute, Hudcova 70,
621 32 Brno,1 and National Institute of
Public Health, Center for Food Chain Hygiene, Palackeho 1-3, 612 42 Brno,2 Czech Republic
Received 27 November 2000/Accepted 14 February 2001
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ABSTRACT |
Retron reverse transcriptases are unusual procaryotic enzymes
capable of synthesis of low-molecular-weight DNA by reverse transcription. All of the so-far-described DNA species synthesized by
retron reverse transcriptases have been identified as multicopy single-stranded DNA. We have shown that Salmonella enterica
serovar Enteritidis is also capable of synthesis of the
low-molecular-weight DNA by retron reverse transcriptase. Surprisingly,
Salmonella serovar Enteritidis-produced
low-molecular-weight DNA was shown to be a double-stranded DNA with
single-stranded overhangs (sdsDNA). The sdsDNA was 72 nucleotides (nt)
long, of which a 38-nt sequence was formed by double-stranded DNA with
19- and 15-nt single-stranded overhangs, respectively. Three open
reading frames (ORFs), encoded by the 4,053-bp plasmid, were essential
for the production of sdsDNA. These included an ORF with an unknown
function, the retron reverse transcriptase, and an ORF encoding the
cold shock protein homologue. This plasmid was also able to confer
phage resistance onto the host cell by a mechanism which was
independent of sdsDNA synthesis.
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INTRODUCTION |
Salmonella enterica
serovar Enteritidis frequently contains plasmids. The best understood
is the serovar-specific plasmid which encodes for virulence genes,
e.g., spv, pef, or rck (6, 12, 29, 30,
39). Besides this plasmid, wild-type Salmonella serovar Enteritidis strains occasionally contain additional plasmids, frequently of low molecular weight. They have been extensively used in
molecular typing (5, 10, 26, 33), but only a few reports
describing their biological functions have appeared. They have been
suspected to influence resistance to antibiotics (34, 36)
and phages (11), and when transformed into
Escherichia coli, these plasmids affect its growth
(16). During our recent studies in molecular typing
(31, 32), we have observed that in a particular serovar
Enteritidis strain, a single low-molecular-weight plasmid was
responsible for the resistance to phage infection. Loss of this plasmid
resulted in conversion of phage type PT21 to phage type PT1
(32). The difference between PT1 and PT21 strains is
mainly in resistances to phages P3, P5, and P7 of the standard phage
typing set, to which strains of PT21 are resistant while strains
belonging to PT1 are sensitive (38). Therefore we
sequenced the whole plasmid, and sequence analysis revealed that it
coded for an open reading frame (ORF) similar to those of the retron
reverse transcriptases (RRT) (35).
RRTs are unusual enzymes that were first described for Myxococcus
xanthus (18) and later for E. coli
(21, 23). They catalyze the synthesis of multicopy
single-stranded DNA (msDNA) (8, 40), which is usually 50 to 150 nucleotides (nt) in length, present freely in the cytoplasm of
bacterial cells (7, 28). Biosynthesis of msDNA by the RRT
has been described in detail using in vitro systems (15),
but the biological role of msDNA is still unknown. RRTs are present in
most of the M. xanthus strains (8, 19) but in
only 10 to 15% of E. coli isolates (14). Genes
for RRT were localized on the chromosomal DNA. In some cases, mainly in
E. coli, the chromosomal loci resembled structures of bacteriophages (9), and some bacteriophages were even
found to encode RRT as well (17). So far, RRT has never
been described to be plasmid encoded. There was only a single report on
its presence in Salmonella (27).
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MATERIALS AND METHODS |
Bacterial strains.
Two field strains of
Salmonella serovar Enteritidis, previously characterized by
plasmid profiling (31, 32) and by phage typing
(38), were used as the donors and final recipients of plasmids. Salmonella serovar Enteritidis strain 2159 was of
phage type PT21 and plasmid type SE55IJ, i.e., it contained the 55-kb virulence plasmid, plasmid I, and plasmid J (31). After
prolonged storage at 4°C (for about a year), Salmonella
serovar Enteritidis strain 2160, originally of the same plasmid
profile, SE55IJ, spontaneously lost the plasmid designated I. This
resulted in a strain of plasmid type SE55J and conversion of phage type
PT21 to phage type PT1 (see Fig. 1). Strains were grown in Luria broth
(Difco) at 37°C in a shaking incubator at 200 rpm.
Sequencing of plasmid I.
Plasmid DNA was purified with a
QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany) from the strain
serovar Enteritidis 2159. Total plasmid DNA was digested with the
restriction endonuclease TaqI and ligated into the
ClaI-digested plasmid pBluescript SK(
). Clones containing
fragments from plasmid I were selected by colony hybridization using
plasmid I, labeled by the ECL Direct Nucleic Acid Labelling and
Detection System (Amersham, Little Chalfont, United Kingdom) in
low-melting-point agarose, as a probe. Six clones were selected for the
initial sequencing. Obtained sequences were used for the design of
walking primers, which were used in sequencing reactions with serovar
Enteritidis 2159 total plasmid DNA as a template. Sequencing was
carried out with an ABI Prism 310 Genetic Analyzer. Sequence alignments
and basic analysis were carried out using Gene Compar software (Applied
Maths, Kortrijk, Belgium). MFold calculation was done with the GCG
software package. Sequence comparison with GenBank entries was done by
basic BLAST (http://www.ncbi.nlm.nih.gov).
Detection and initial characterization of low-molecular-weight
DNA.
The DNA produced by RRT was isolated as described previously
(3, 19). During the purification procedure, RNase A (100 µg/ml) was present in the cell resuspension buffer, unless otherwise stated. After electrophoresis on a 10% polyacrylamide gel, the DNA was
visualized by staining with Sybr Gold fluorescent dye (Molecular
Probes). Prior to being electroblotted onto a nylon membrane (Hybond N;
Amersham), the samples were denatured in 0.25 M NaOH for 15 min. Using
a PCR Master Mix kit (Qiagen), a PCR product (658 bp in size) spanning
the expected low-molecular-weight DNA locus was amplified, labeled by
the ECL Direct Nucleic Acid Labelling kit, and used as a probe in
hybridization. Sequences of the primers used for the probe
amplification were as follows: P2, 5' AAT TAT CCT GAG TGC CGA TG
3'; P3, 5' TAA ACC TGG GTT TAT TCA TG 3'.
To determine the nature of the low-molecular-weight DNA, it was treated
with 10 µg of RNase A/ml for 30 min at 37°C, 10 µg of DNase I/ml
for 30 min at 37°C, 20 U of S1 nuclease for 30 min at 23°C, or 10 U
of RNase H for 30 min at 23°C. The low-molecular-weight DNA was also
tested for heat resistance for 10 min at 99°C in a thermocycler.
Sequence characterization of low-molecular-weight DNA.
Two
independent protocols were used for sequence determination. First, the
low-molecular-weight DNA was used as an in vivo-produced primer in a
PCR to which only one standard PCR primer was added. Primers of both
possible orientations were tested in these PCRs. Two selected PCR
products were cloned into pcDNA3.1/V5/His-TOPO (Invitrogen),
recombinant plasmids were purified, and the sequence of the cloned PCR
product was determined. The same protocol was used for sequence
determination of the S1 nuclease-treated low-molecular-weight DNA.
In a second protocol, the low-molecular-weight DNA was excised and
extracted from a polyacrylamide gel using the QIAEX II
Gel Extraction
kit (Qiagen), incubated with
Taq polymerase for
20 min at
72°C to add 3'-end A overhangs, and cloned into the
pCR4 TOPO plasmid
(Invitrogen). The sequence of the cloned DNA
was determined by
sequencing with standard M13 forward and reverse
primers.
Identification of genes essential for the synthesis of
low-molecular-weight DNA.
Two independent protocols were used. In
the first protocol, two fragments of plasmid I were PCR amplified. The
forward primer in both reactions was identical and was located 795 bp
upstream from the start codon of retron reverse transcriptase. The
reverse primer in the first kind of PCR allowed amplification of the
low-molecular-weight DNA locus together with retron reverse
transcriptase. The second reverse primer allowed amplification of the
above-mentioned region, including an ORF5 downstream from the retron
reverse transcriptase. Amplification products were cloned into
pcDNA3.1/V5/His-TOPO and transformed into host E. coli TOP10
(provided with the cloning kit by Invitrogen). After multiplication in
E. coli, the recombinant plasmids (pRT7 where ORF5 was
missing and pRT21 including the ORF5) were purified and
electrotransformed (E. coli Pulser; Bio-Rad) into Salmonella
serovar Enteritidis 2160 to check for restoration of
low-molecular-weight DNA synthesis and also for the restoration of the
original phage type.
In a second experiment, insertional mutagenesis was applied. An
ampicillin resistance gene cassette was prepared by amplification
of
the
bla gene from the pBluescript plasmid with the following
primers: AmpF, 5' GTT AAG GGA TTT TGG TCA TG 3'; AmpR,
5' GCA
CTT TTC GGG GAA ATG TG 3'.
Four pairs of these primers differed in the modifications of their 5'
ends by the presence of either the
NsiI,
EcoRI,
SacI,
or
EcoRV restriction sites, respectively.
The 1,092-bp PCR products
were purified with the QIAquick Gel
Extraction kit (Qiagen), digested
with the appropriate restriction
endonuclease, purified with the
Gel Extraction kit again, and cloned
into the plasmid I, digested
and purified in the same way with only one
exception-the
EcoRV
blunt-ended PCR product was cloned into
the
SfcI site of the plasmid
I, which was filled by Klenow
fragment to produce a blunt-ended
linear plasmid molecule. Ligation
mixtures were used for electrotransformation
of the
Salmonella serovar Enteritidis 2160 strain, selecting for
ampicillin-resistant colonies. Clones containing plasmid I with
the
inserted ampicillin resistance gene cassette were selected
by brief
phenol plasmid extraction (
1) followed by PCR
verification.
Nucleotide sequence accession number.
The complete sequence
of the plasmid I encoding RRT is available in GenBank under accession
number AF218051.
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RESULTS |
Sequencing of plasmid I.
We have determined the complete
sequence of the low-molecular-weight plasmid of Salmonella
serovar Enteritidis which was capable of protecting the bacterium
against phage infection. The original field strain containing the
plasmid was of phage type PT21; after the spontaneous loss of this
plasmid, the resulting strain was of phage type PT1 (Fig.
1). The size of the plasmid is 4,053 bp, and it codes for five possible ORFs (Fig.
2).
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FIG. 1.
Plasmid profiles of Salmonella serovar
Enteritidis 2159 (lane IJ) and serovar Enteritidis 2160 (lane J) after
electrophoresis on an 0.8% agarose gel. Plasmid I is marked with an
asterisk. A 1-kb ladder was used as the molecular size standard.
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ORF1 encoded a protein (12.2 kDa) showing homology to ORFs already
identified in similar-sized plasmids of
E. coli and
Salmonella enterica serovar Typhimurium; however, its
function was not determined.
ORF3 (6.8 kDa) was of a sequence which did
not match any sequence
available in GenBank. For the remaining three
ORFs, the function
could be predicted based on the similarities to
proteins with
already-identified functions. ORF2 was homologous to
mobA, a protein
involved in the initiation and termination
of conjugal DNA transfer
(
2); ORF4 shared homology with
RRTs of
E. coli (for retron
Ec107, 30% identity and 52%
similarity; for Ec86, 27% identity
and 48% similarity; and for Ec67,
27% identity and 46% similarity)
and also with the
M. xanthus retron Mx65 (23% identity; 42% similarity).
ORF5 was
similar to cold-shock proteins from multiple bacterial
species, such as
Pseudomonas aeruginosa, Lactobacillus plantarum, Staphylococcus
aureus, Listeria monocytogenes, or
Bacillus subtilis,
displaying identities and similarities around 35 and 55%, respectively
ORF1 and ORF2 were located in the part of the plasmid which showed
extensive homology to sequences of other low-molecular-weight
plasmids
and were therefore quite probably linked with the replication
and
maintenance of the plasmid I in the bacterial cell (position,
bp 1 to
1600). ORFs 3, 4, and 5 were located in the remaining
part of the
plasmid. Since we did not expect that phage resistance
could be
influenced by ORF1 and ORF2, the remaining three ORFs
were analyzed
further in
detail.
Detection and initial characterization of low-molecular-weight
DNA.
The products of almost all RRTs described so far were
multicopy single-stranded DNAs (14, 19, 27, 28). As
expected, similar low-molecular-weight DNA was present in the
plasmid-containing Salmonella serovar Enteritidis 2159 strain but was missing in the Salmonella serovar Enteritidis
2160 strain (Fig. 3). The DNA was
estimated to be approximately 82 nt in length. The DNA was resistant to
RNase A and RNase H treatment. On the other hand, it was sensitive to
DNase I and partially sensitive to S1 nuclease treatment (Fig.
4). Treatment with S1 nuclease resulted
in an increase in polyacrylamide gel electrophoresis mobility,
corresponding to an approximately 20-bp decrease in molecular size. The
results therefore indicated that the product of RRT was either a
single-stranded DNA with a complex secondary structure or a
double-stranded DNA species with a single-stranded overhang(s) at the
end(s) of the molecule. Such results were obtained for the
low-molecular-weight DNA purified with the protocol in which the RNase
A was present in first cell resuspension buffer. In a second
experiment, we purified the low-molecular-weight DNA by the same
protocol, however, without RNase A. As can be seen in Fig.
5, the DNA was present, although in lower
quantities. The quantity of the low-molecular-weight DNA was increased
considerably after RNase A treatment. However, the other enzymes had
standard effects on this sample
S1 nuclease treatment resulted in
increased mobility, and DNase I treatment considerably decreased the
amount of the low-molecular-weight DNA (Fig. 5).
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FIG. 3.
Low-molecular-weight DNA produced by
Salmonella serovar Enteritidis 2159 (lane IJ) and
Salmonella serovar Enteritidis 2160 (lane J) after
electrophoresis on a 10% polyacrylamide gel. A 20-bp ladder was used
as a molecular size standard.
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FIG. 4.
Low-molecular-weight DNA electrophoresed on a 10%
polyacrylamide gel after treatment with different enzymes. Lane 1, a
20-bp ladder used as a molecular size standard; lane 2, untreated
control DNA; lane 3, DNA treated with RNase H; lane 4, heat-treated
DNA; lane 5, DNA treated with RNase A; lane 6, DNA treated with DNase
I; lane 7, DNA treated with S1 nuclease.
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FIG. 5.
(A) Low-molecular-weight DNA isolated in the absence of
RNase A in the purification protocol, treated with different enzymes,
and electrophoresed on a 10% polyacrylamide gel. Lane M, a 20-bp
ladder used as a molecular size standard; lane 1, control DNA purified
in the presence of RNase A; lane 2, untreated DNA; lane 3, DNA treated
with RNase H; lane 4, heat-treated DNA; lane 5, DNA treated with RNase
A; lane 6, DNA treated with DNase I; lane 7, DNA treated with S1
nuclease. (B) Since the presence of low-molecular-weight DNA is hidden
by the presence of RNA, hybridization was carried out to detect the
low-molecular-weight DNA.
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Sequence characterization of low-molecular-weight DNA.
To
identify the sequence of the low-molecular-weight DNA and to localize
it on the plasmid map, we used the approximately 82-nt DNA as one of
the primers in PCR together with only a single in vitro-synthesized
primer. Surprisingly, the PCRs repeatedly resulted in a positive
amplification regardless of the orientation of the in vitro-synthesized
primer added to the reaction. The PCRs were positive even in the
presence of RNase A in the PCR tube (not shown). Two PCR products, each
originating from oppositely oriented PCRs, were cloned into
pcDNA3.1/V5/His-TOPO and sequenced. Comparison of both sequences
revealed an overlap of 72 bp which must have served as an in vivo
primer in PCRs and therefore represented the sequence of
low-molecular-weight DNA. Exactly the same protocol was applied to
samples which were first treated with S1 nuclease. After cloning and
sequencing of two PCR products, the overlap was only 38 bp in size and
this sequence was internal to the 72-bp sequence of nontreated sample.
The sequence of the full-size low-molecular-weight DNA was
independently confirmed by its direct cloning after
Taq
polymerase
extension. In this experiment, the identified 72-bp sequence
was
identical to the sequence determined by the PCR-based protocol.
All
of this led us to the conclusion that the low-molecular-weight
DNA
synthesized by serovar Enteritidis is a small double-stranded
DNA
(sdsDNA) with single-stranded overhangs (Fig.
6).
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FIG. 6.
Expected structure of sdsDNA. The total length was
determined to be 72 nt, with a central double-stranded core of 38 bp
and 19- or 15-nt-long single-stranded overhangs.
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Sequence analysis of the locus encoding sdsDNA.
Sequence
analysis of the part of the plasmid encoding the RRT and sdsDNA
revealed a 13-bp-long inverted repeat which started 3 nt downstream
from the sdsDNA (positions 2306 to 2318 included), and the
complementary repeat (positions 2578 to 2590 included) ended 6 bp
upstream of the start codon of the RRT. The loop formed by the inverted
repeat contained the whole ORF3, the termination codon of which was
located 2 bp upstream of the start of the inverted repeat (Fig.
7). This loop was also predicted by the
MFold calculation (not shown).
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FIG. 7.
Detailed description of RRT locus. Primers P1 and P4
were used together with low-molecular-weight DNA in PCRs. Finally,
amplification products originating from P1-sdsDNA and P4-sdsDNA PCRs
were cloned and sequenced. The P1-P5 and P1-P6 primer pairs were used
in the cloning of the DNA fragments necessary for sdsDNA production and
phage resistance. Cloning of the P1-P5 PCR product resulted in the pRT7
plasmid, and cloning of the P1-P6 PCR product resulted in the pRT21
plasmid. MFold calculation confirmed the predicted ORF3 loop.
Calculation was performed on the sequence starting from the position
2000 of the plasmid and ending at bp 3000. The figure illustrates the
ORF3 predicted loop.
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Identification of genes essential for the sdsDNA production and
phage resistance.
Finally we verified the function of ORF3, ORF4
(rrt), and ORF5 (csp) in the production of the
sdsDNA and in the resistance to phage infection. Using PCR, two
different fragments of plasmid I were amplified and cloned to form
plasmids pRT7 and pRT21. Plasmid pRT7 contained the sdsDNA region,
ORF3, and ORF4, and plasmid pRT21 contained the same genes and also
ORF5. Only plasmid pRT21, which contained the sdsDNA region together
with RRT and the ORF5 located downstream of the RRT, allowed the
synthesis of sdsDNA in Salmonella serovar Enteritidis 2160. However, these plasmids (pRT7 and pRT21) were quite unstable at 37°C
in Salmonella serovar Enteritidis 2160 in the absence of
ampicillin and made the phage typing difficult to assay.
Therefore we performed insertional mutagenesis on plasmid I, which
enabled a proper investigation of the role of individual
ORFs in the
biosynthesis of the sdsDNA and also in phage resistance.
An ampicillin
resistance gene cassette was inserted into the
NsiI
site
where no ORF was predicted, into the
SacI site to interrupt
the ORF3 sequence, into the
EcoRI site to interrupt the RRT,
and
into the
SfcI site of the plasmid to inactivate the
cold-shock
protein homologue (Fig.
2). When these plasmids were
transformed
into
Salmonella serovar Enteritidis 2160, the
sdsDNA synthesis
was restored only when the ampicillin resistance gene
cassette
was inserted in the
NsiI site, outside all the
predicted ORFs.
The original phage type, PT21, was not restored when
the ampicillin
resistance gene cassette was inserted in the
SacI site in the
ORF3 (Table
1). The remaining three recombinant
plasmids restored
phage resistance to the host strain,
Salmonella serovar Enteritidis
2160, although plasmid I with
the ampicillin resistance cassette
inserted in the
NsiI site
gave sometimes ambiguous results in
phage typing.
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TABLE 1.
List of Salmonella strains used in the study
and their abilities to synthesize sdsDNA and to resist phage infection
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DISCUSSION |
In the present study we report on the low-molecular-weight plasmid
which encodes the RRT and influences resistance to phage infection in
Salmonella serovar Enteritidis. RRTs are quite ubiquitous in
Myxococcus spp. (8, 19) and rather rare in
field strains of E. coli and Salmonella
(14, 27). They have been reported to be chromosomally
encoded or phage encoded. Here the first evidence for plasmid-encoded
reverse transcriptase is presented. Furthermore, we have shown that the
enzyme is encoded by a very small plasmid, the size of which was only
4,053 bp.
Most of the so-far-described RRTs catalyze synthesis of msDNA. Our
results surprisingly show that the DNA produced by the RRT of serovar
Enteritidis is double-stranded DNA with single-stranded overhangs.
Because RNase A treatment of natural sdsDNA repeatedly resulted in an
increase in sdsDNA quantity, it seems probable that during its
biosynthesis it is bound to an RNA molecule. However, this RNA molecule
must be of a greater molecular size, definitively more than several
hundred nucleotides, since we should have detected molecules below this
limit after blotting and hybridization (Fig. 5). The sdsDNA was also
free of any DNA-RNA hybrid molecule, since the sdsDNA was totally
resistant to RNase H treatment. S1 nuclease treatment, on the other
hand, always increased the mobility of sdsDNA in polyacrylamide gel
electrophoresis, indicating that single-stranded structures are present
in the molecule. We found that the sdsDNA was efficiently used by
Taq polymerase as a primer in PCR and, consistent with the
suggested model of double-stranded DNA, the sdsDNA could prime the PCR
in both possible directions. Successful direct cloning of the sdsDNA
after incubation with Taq polymerase indicates that the
single-stranded overhangs are 5' ends of both strands of the
moleculeonly in this case, the 3' recessing ends could be efficiently
used by Taq polymerase as a template. This could be also a
reason that we did not succeed in direct cloning of the sdsDNA treated
with the S1 nucleasealready blunt-ended sdsDNA was probably not as
effective a template for the Taq polymerase as the molecule
with 3' recessing ends. We succeeded, however, in amplifying, cloning,
and sequencing the PCR products derived from the PCR in which sdsDNA
treated with S1 nuclease and external primers of both possible
orientations were used. Comparison of the sequences of the two PCR
products allowed us to identify an overlap of 38 bp, which represented the S1 nuclease treatment-resistant part of the sdsDNA. Double-stranded DNA with 5'-end single-stranded overhangs has not been reported so far
to be a product of RRT. Although most of the reports describe msDNA as
a single-stranded DNA bound by an unusual 2'-5' phosphodiester bond to
RNA (15, 27), there are studies reporting that the final
product could be RNA-free single-stranded DNA (24), and at
least one study on in vitro synthesis showed that RRT is capable of
synthesis of double-stranded DNA molecules (22).
The RRT of Salmonella serovar Enteritidis possessed
additional unique properties. The RRT itself was not enough to initiate the production of sdsDNA. A functional, downstream-located sequence coding for a peptide of 86 amino acids homologous to cold-shock proteins was necessary for the production of the sdsDNA. In M. xanthus, the msDNA was shown to form a complex with multiple
proteins in the cell (37), and so it is tempting to
speculate on the similar role of csp in the biosynthesis of
the sdsDNA, although it might be also possible that the mere insertion
of an approximately 1-kb sequence of the Ampr gene cassette
influences the secondary structure of the RNA transcript to an extent
incompatible with the reverse transcription. Next, the structure of the
whole locus was different from that described in previous reports on
RRTs. The inverted repeats a1 and a2 (15, 18, 23), 13 bp
long in this case, could be identified, but the sdsDNA coding region
was located not inside but outside the loop formed by the inverted
repeats. Instead, inside the loop formed by the a1 and a2 inverted
repeats, an additional ORF3, of no homology to GenBank entries, was located.
Plasmid I was the first identified as being linked with resistance to
phage infection. Strains bearing this plasmid were of phage type PT21,
while a strain which spontaneously lost this plasmid was of phage type
PT1. The difference between PT1 and PT21 strains is mainly in
resistances to the phages P3, P5, and P7, to which strains of the PT21
group are resistant and strains belonging to the PT1 group are
sensitive (38). Surprisingly, the resistance to phage
infection was independent of the sdsDNA production, and it was also
independent of functional ORF4 (rrt) and ORF5
(csp). It was, on the other hand, absolutely dependent on
functional ORF3. Insertion upstream of the ORF3 into the
NsiI site, although it did not influence the sdsDNA
synthesis, gave ambiguous results in phage resistance (Table 1). We
therefore conclude that the phage resistance and sdsDNA production do
not correlate. How the phage resistance is expressed and the sdsDNA is
synthesized are currently unknown; however, we speculate that the 13-bp
inverted repeats and the loop structure containing the ORF3 are of
particular importance. Formation of such a loop on the mRNA level could
lead to a single gene translation of ORF3 and could also put the sdsDNA
locus in a close proximity to the rrt gene.
The biological role of msDNA in Myxococcus and E. coli remains unclear (28). It was considered to be
mutagenic (4, 20, 25), and it was also shown to be induced
in stationary phage (13). In our study, we have shown that
the plasmid I encodes the RRT and the presence of the plasmid
correlates with the synthesis of sdsDNA. The plasmid is also involved
in resistance to phage infection. Plasmid I therefore represents a very
interesting, self-replicating model of a DNA molecule encoding the RRT
with multiple unique features.
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ACKNOWLEDGMENTS |
This work has been supported by grants EP6076 and QC0195 from the
Ministry of Agriculture of the Czech Republic.
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FOOTNOTES |
*
Corresponding author. Mailing address: Veterinary
Research Institute, Hudcova 70, 621 32 Brno, Czech Republic. Phone:
420-5-41321241. Fax: 420-5-41211229. E-mail: rychlik{at}vri.cz.
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Journal of Bacteriology, May 2001, p. 2852-2858, Vol. 183, No. 9
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.9.2852-2858.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
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