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Applied and Environmental Microbiology, August 1999, p. 3582-3587, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Use of Single-Strand Conformation Polymorphism
Analysis To Examine the Variability of the rpoS
Sequence in Environmental Isolates of Salmonellae
Suzanne J.
Jordan,1
Christine E. R.
Dodd,1,* and
Gordon S. A. B.
Stewart2,
Division of Food Sciences, School of
Biological Sciences, University of Nottingham, Sutton Bonington Campus,
Loughborough, Leicestershire LE12 5RD,1 and
School of Pharmaceutical Sciences, University of
Nottingham, University Park, Nottingham NG7
2RD,2 United Kingdom
Received 29 January 1999/Accepted 27 May 1999
 |
ABSTRACT |
The natural environment places its resident microflora under
stress, which may often result in adaptation by the microflora in order
to increase the probability of survival. One such mechanism that has
been postulated involves rpoS, which encodes a sigma factor
that is known to enhance survival upon exposure to stress. The present
work aimed to examine the genetic variability of rpoS in a
selection of Salmonella enterica subspecies environmental isolates with an automated single-strand conformation polymorphism analysis technique. The results indicated that sequence variation does
occur and that these changes are mainly located in two areas: at the
center and near the end of the coding region. The variability was
generally at the single-base level, although one strain (S. arizonae) did demonstrate significant differences in nucleotide sequence.
| |
INTRODUCTION |
RpoS (
S,
38) is a key element for the survival of several
gram-negative bacteria (including the salmonellae, Escherichia
coli, pseudomonads, and Vibrio spp.) in adverse
situations, e.g., entry into stationary phase (11, 21) and
exposure to sublethal stresses such as osmotic shock (12)
and low pH (23). The protein aids transcription of at least
30 genes and operons that form part of the bacterial defense
(5) and virulence (8) mechanisms. Alignment of
RpoS with RpoD (70) (25) suggests that
functional regions present are conserved in core binding (to the core
polymerase), in the "RpoD box" (implicated in DNA strand opening),
and in the 
10 and 35 binding sites (for promoter recognition).
Over the last few years, isolates responsible for outbreaks of
food-borne salmonellosis, e.g., Salmonella typhimurium DT104 (38), have been reported to be increasing their ranges of
antibiotic resistance, and further work has indicated that some
E. coli and Salmonella sp. isolates are capable
of entering a hypermutatable state (observed as a gain in resistance to
several antibiotics [22, 37]). Most food products
create a relatively harsh microenvironment, resulting in the resident
flora being in stationary phase (34) due to the levels of
stress experienced in this situation. Recent reviews suggest that the
preservative techniques utilized by the food industry to control and/or
reduce the microbial load within these products may actually facilitate
the evolution of more-resistant food pathogens (1, 20). One
of the problems in improving food safety is the potential for
rpoS variability between different isolates of the same
strain, which occurs both in the laboratory and in natural environments
(17, 40, 41). In certain circumstances, this variation has
been thought to confer a selective advantage on individual bacteria
within a population experiencing nutrient deprivation (41).
In previous work, screening for mutations of rpoS has been
approached by identifying strains demonstrating phenotypic differences and then sequencing the gene (39, 41); however, this
technique does not detect silent mutations. The use of genetic
screening allows all nucleotide differences to be located; one
technique that is capable of performing this task is single-strand
conformation polymorphism analysis (SSCPA) (19). This method
relies on electrophoretic separation of fragments according to their
sizes and secondary DNA structures, which are sequence dependent
(26, 33). Multiple fluorescence-based PCR-SSCPA (6,
15) has provided the capacity for simultaneous labelling of
several fragments as well as the potential for an automated data
collection system (4).
The present work aimed to evaluate the level of nucleotide variation in
rpoS in the salmonellae. A selection of 18 environmental and
human isolates of predominantly Salmonella enterica
subspecies were screened along the entire length of the rpoS
gene by SSCPA, with potential changes being confirmed by sequence analysis.
| |
MATERIALS AND METHODS |
Strains.
A list of strains utilized is shown in Table
1.
Primer design for PCR fluorescent labelling.
Primer
sequences together with their fluorescent labels are shown in Table
2. In designing the primers, two major
points were considered: (i) the optimal length for detection of
single-base-pair changes is 200 bp (10), and (ii) changes at
the end of the fragment are often more difficult to detect than those
at the center (36). The six regions (A to F) covered the
whole coding region of the gene, together with a small upstream region
to ensure that the transcriptional start site was included. The primers
were designed by utilizing Primer Express software, version 1.0 (Perkin-Elmer Applied Biosystems, Foster City, Calif.), with the four
available sequences (GenBank) for rpoS in salmonellae
(accession numbers are given in parentheses): S. typhi
(X81641), S. enterica (X82129), and S. typhimurium (U05011 and X77752). The numbering system employed
used the base pair numbering of S. typhi, which, as the longest sequence (1,707 bp [35]), avoided confusion
over base pair locations within rpoS. The area to be
screened (Fig. 1) starts just upstream of
the transcriptional start site, which, according to published data
(35), is at bp 423.
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FIG. 1.
rpoS primers utilized for SSCPA of
rpoS and predicted functional regions within RpoS (taken
from reference 25). In the upper section, regions of
function within RpoS are highlighted on the protein along with their
locations. The lower section shows the rpoS gene and the
locations of the primers utilized for the screening process. aa, amino
acids.
|
|
PCR amplification of rpoS regions.
Cultures were
streaked onto Luria (Lennox) agar (Difco, Surrey, United Kingdom) and
incubated overnight at 37°C. The template was prepared for PCR by
emulsifying a single colony from the overnight culture in 100 µl of
sterile reverse-osmosis (RO) water. Reaction mixtures of 50 µl,
containing 16 pmol of primers (Perkin-Elmer Applied Biosystems), 25 mM
(each) deoxynucleoside triphosphates (Pharmacia Biotech, Herts, United
Kingdom), 1 mM MgCl2 (Advanced Biotechnologies,
Leatherhead, United Kingdom), and 4.6 µl of buffer IV (200 mM
[NH4]2SO4, 750 mM Tris-HCl [pH
9.0] at 25°C, and 0.1% [wt/vol] Tween) supplied with
Taq DNA polymerase AB0289 (Advanced Biotechnologies), were
prepared and made up to volume with sterile RO water. Following a hot
start of 10 min at 95°C and subsequent addition of 1 U of
Taq DNA polymerase (Advanced Biotechnologies), PCR was
performed with 35 cycles of 95°C for 15 s, 55°C for 30 s,
and 72°C for 30 s and a final cycle of 72°C for 10 min
(Progene [FPROG050]; Techne [Cambridge] Ltd., Cambs, United
Kingdom). Correct amplification was verified by electrophoresing the
products together with 1.5 µg of a 100-bp ladder (Pharmacia Biotech)
on a 1.5% (wt/vol) agarose gel (NuSieve 3:1; FMC, Kent, United
Kingdom) containing 1 µl of a solution containing 100 mg of ethidium
bromide (Sigma, Dorset, United Kingdom) ml
1.
SSCPA of rpoS.
Optimization of all conditions for
SSCPA was carried out by using paired strains with known
single-base-pair changes in a particular region.
The labelled PCR products were purified with a PCR purification kit
(Qiagen, West Sussex, United Kingdom) according to recommended
instructions (
32), and the DNA was eluted in 50 µl of
sterile
RO water. A sample dilution (ranging from 1:2 to 1:30) was
performed
as previously described (
16). From the resulting
dilution, 5
µl was added to 0.5 µl of 0.1 M NaOH, which was heated
at 95°C
for 5 min and immediately snap-cooled on ice. After
denaturation,
0.5 µl of Genescan-500 (TAMRA) (Perkin-Elmer
Applied Biosystems)
was added to each sample to act as the internal
lane standard.
The samples (for regions A to E) were loaded onto a
0.4-mm prechilled
10% (wt/vol) polyacrylamide gel (12.5 ml of Ultra
Pure electrophoresis-grade
40% [wt/vol] acrylamide-bis acrylamide
[37.5:1] [Sigma], 5 ml
of 10× TBE buffer [890 mM Tris-borate, 890 mM boric acid, and
20 mM EDTA {Sigma Ultra Pure} at a pH of 8.3 at
ambient temperature],
and 32.5 ml of distilled water prepared as
previously described
[
29]). Region F samples required
a lower-percentage gel (8%
[wt/vol]).
Electrophoresis was performed in prechilled 1× TBE buffer at 500 V for
20 h in a 373 DNA Sequencer (Perkin-Elmer Applied Biosystems);
buffer was kept chilled for the first 30 min of the run time by
the
periodic addition of miniature sealed ice blocks. Data were
collected
and analyzed with Genescan 672 software (version 1.2.1)
(Perkin-Elmer
Applied Biosystems). Relative mobility calibration
was constructed by
utilizing the second-order least squares curve
(i.e., linear
regression) to provide the best interlane comparison.
Internal lane
standard peaks were allotted relative mobility values
(scan number
divided by 10) in order to provide a numerical comparison
of fragment
mobility between
lanes.
Sequence analysis.
Sequencing was performed with a 373 DNA
sequencer (Perkin-Elmer Applied Biosystems) with the Taq Dye
Deoxy terminator cycle sequencing kit (Perkin-Elmer Applied Biosystems)
and the relevant reverse SSCPA primers (A to F) as required.
| |
RESULTS |
Calculating gel variation of a "standard sequence."
SSCPA
relies on detection of differences in relative mobility during
electrophoresis between PCR products of the same region. Because of gel
variation, the same sequence run on different occasions may give
differences in relative mobility. To allow changes in nucleotide
sequence to be determined, this intrinsic variation in the relative
mobility of a given sequence must first be estimated. For each region,
a known sequence was taken as standard ("standard sequence"). PCR
products of this standard sequence were run in five separate wells of
duplicate gels, and a mean and standard deviation of relative mobility
for each gel for the standard sequence of each region was calculated.
Table 3 shows the duplicate means and
standard deviations [(n1)] of relative
mobility for each standard sequence from all of the regions within
rpoS (A to F). The standard deviations of the five samples
of identical sequence varied slightly between duplicate gels, with
region D showing the largest discrepancy. This suggests that comparison between gels is possible, although it is not advisable for complete accuracy in the prediction of sequence variability. The variations in
standard deviation between regions ranged from 0.3 for region E to 1.54 for region D. Therefore, to ensure that a change in relative mobility
reflected true sequence variation from the standard sequence, a value
of more than 2 standard deviations from the mean for a region was taken
as a threshold value with defined confidence limits of 95%.
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TABLE 3.
Calculated standard deviations and means of relative
mobility of a single sample for the six regions
of rpoSa
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|
Detection of nucleotide differences in rpoS.
PCR
products for the six regions of the 18 strains of Salmonella
were analyzed by SSCPA, and sequence variations were determined by
comparison of their relative mobilities with those of the standard sequences. These 18 strains included some isolates where
single-base-pair changes were known from previous sequencing work to
demonstrate single-base-pair changes in one of the regions. Those
strains for which the relative mobilities for a region were outside the threshold value of 2 standard deviations are shown in Table
4. Three of the rpoS regions
(A, D, and E) showed no variation in sequence, as indicated by the lack
of shifts in fragment relative mobilities, and these were not analyzed
further. Of the regions where strains demonstrated potential nucleotide
differences (B, C, and F), C had the greatest level of variation. No
strains showed variation in more than one region. The degree of shift
was variable, suggesting that the type and extent of discrepancy may be
strain dependent. In particular, the shifts with S. arizonae
in region F seemed to be significantly larger than in other strains.
Although S. amina was already known to contain a
single-base-pair change (C to T) in region B (from prior sequence
analysis) (Table 5), the shift in
relative mobility fell inside the 2-standard-deviation threshold and
therefore was not detectable by this method.
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TABLE 4.
Relative mobilities for the six regions of
rpoS from Salmonella environmental isolates
demonstrating relative mobility shifts
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|
For
S. arizonae, primer pair C failed to amplify a fragment
with the cycle parameters described above; therefore, each region
C
primer was used with a primer from another region. The primer
combination A forward and C reverse was successful in producing
a PCR
product, but the combination C forward and E reverse was
not. These
products were analyzed for sequence variation as described
below.
Sequence analysis of variant strains.
Confirmation of
potential nucleotide changes was verified with sequence analysis by
using duplicate PCR amplifications containing the region of interest.
In most cases, only a single-base-pair change was responsible for the
shift in fragment migration (Table 5). As expected, greater shifts in
mobility were seen with region C. Of the three sites where changes were
evident, one was consistent: a T-to-C change at bp 897 seen in S. amsterdam and S. typhimurium.
While the location of the change in region B of
S. bovis
morbificans has been identified, the exact sequence change has yet
to be determined: sequence data suggest that the population is
a
mixture of G and T at this site, the latter representing a sequence
change.
The increased fragment migration time in
S. arizonae region
F samples was a result of 14 nucleotide changes (Fig.
2) comprising
12 single-base-pair changes
and two insertions. Sequence data
for the amplified product for region
C revealed that a sequence
complementary to the C forward primer
binding region was present
in
S. arizonae. Failure of PCR
with primer set C was postulated
to be due to interference resulting
from a more stable DNA secondary
structure; PCR with increased binding
and denaturation times resulted
in weak amplification of this region.
Addition of dimethyl sulfoxide
(5%) to the reaction mixture gave a
result equivalent to the PCR
results for the other strains. These
results support the hypothesis
that secondary structure interfered with
amplification of region
C in this strain.
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FIG. 2.
Alignment of S. arizonae rpoS with the
published sequence of S. typhi rpoS to show differences in
region F. Nucleotide changes are highlighted in black.
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|
Region C of
S. arizonae also showed significant variation,
with 6 base changes evident (one A-to-T change, three C-to-T changes,
and two T-to-C changes, as shown in Fig.
3). None of these corresponded
to the
changes seen in other strains.
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FIG. 3.
Alignment of S. arizonae rpoS with the
published sequence of S. typhi rpoS to show differences in
region C. Nucleotide changes are highlighted in black.
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| |
DISCUSSION |
Reproducibility of SSCPA.
In this study a threshold value for
relative mobility of 2 standard deviations from the mean of five
replicates of a standard sequence was set as the limit for determining
changes in base sequence. In the majority of cases (five of six), this
arbitrarily chosen value was able to discriminate between fragments
with known single-base-pair differences. Variation in relative
mobilities produced by identical sequences has been attributed to a
number of factors. Previous work has shown that some single-stranded DNA is capable of producing two different conformations
(15), and this would result in differences in mobility for
identical sequences; in some instances both conformations can be
present and run as separate bands in the same lane. Reproducibility of results has also been linked to constant run conditions, including power and temperature (7). The automated analysis system
used here allows conditions to be recorded, enabling comparison between electrophoresis runs for voltage, temperature, and laser current. Temperature variation of the system here was minimized through the use
of prechilled gels and buffer for electrophoresis. These changes
limited the degree of reannealing following denaturation, especially
during the period prior to DNA strand separation in the gel after the
samples had been loaded onto the wells. The stability of
single-stranded DNA has been verified as being temperature dependent
(15, 28), with ambient temperature facilitating reannealing.
Without prechilling, the level of single-stranded DNA visualized by the
laser detection system employed by the 373 DNA sequencer was
undetectable when small fragments (i.e., approximately 100 bp) were run
(results not shown).
However, one base pair variation in region B of the
S. amina
strain (C-to-T change at bp 686) was not always detected by this
technique. One possible explanation for this is the incorporation
of
errors during PCR (
2,
13,
27,
31). Published literature
suggests that the fidelity of a proofreading DNA polymerase is
able to
reduce the level of nucleotide misincorporation by a factor
of 10 (
30); in this instance, however, use of a polymerase with
proofreading capacity (
Ultma DNA polymerase; Perkin-Elmer
Applied
Biosystems) amplified a fragment of identical sequence. The
likely
reason for this observation is the relatively short length of
DNA amplified in each instance and optimization of the cycling
parameters and reagents for
PCR.
Previous work with SSCPA has resulted in a wide range of detection
rates, ranging from 50 to nearly 100% (
4), which can
be
enhanced by the utilization of more than one set of electrophoretic
conditions (
9). The sensitivity of the technique relies
greatly
on the sequence change to influence fragment mobility
(
33),
and in order to do this, it must cause a disruption in
the folding
of the DNA fragment. In certain instances, C-to-T changes
have
not been readily detected (
36), which suggests that
they are
not in regions of single-stranded DNA that directly influence
its secondary structure. Both cytidine and thymidine are pyramidine
bases consisting of a single six-membered ring and therefore will
not
exert as much steric hindrance to single-stranded DNA folding
as the
purines, which comprise a five- and a six-membered
ring.
Analysis of strains.
Of the 18 isolates analyzed by SSCPA, 6 of them contained discrepancies in the nucleotide sequence, as
confirmed by sequence analysis. The level of variation ranged from a
single base pair change to several substitutions, in the case of
S. arizonae. The latter was to be expected, as this group of
strains is reported to be a distant relative of S. enterica
subspecies in terms of biochemical (24) and genetic
(3) classification strategies. The lack of binding of the C
forward primer to the totally complementary binding site in this strain
indicates that the secondary structure of this region may be
interfering with primer binding. This was confirmed by a weak
amplification when the denaturing time was doubled and by a level of
amplification similar to other S. enterica isolates in this
region upon addition of dimethyl sulfoxide to the PCR mixture.
The locations of the nucleotide changes found indicate that certain
areas of the gene (A, D, and E) remain conserved within
the subspecies
of
S. enterica. These areas may be of specific
importance to
the cell, such as regions coding for protein functionality
or turnover.
RpoS is an important cell constituent which facilitates
survival under
adverse conditions; therefore, it is to be expected
that some sections
of the gene may be conserved. Alignment with
other sigma factors with
known functional regions (
25) indicates
that regions C and
F, which showed the greatest variation, may
be involved in binding to
the core polymerase and the
35 binding
site, respectively (Fig.
1).
The predominant type of nucleotide
variation in this work is a single
substitution or insertion along
the sequence, which is similar to
previous work published for
rpoS from both laboratory and
environmental isolates of
E. coli (
40,
41). Not
all mutations are beneficial to the bacterial
population, and in most
instances they lead to a reduced ability
to survive (
18,
40). However, there have been reported incidences
of mutations in
rpoS which enhance survival in long-term storage,
as defined
by the GASPing phenotype (
41). These mutations are
not
necessarily imperative for survival but have been postulated
to provide
individuals with a competitive edge. The role of the
mutations detected
in the present work is yet to be discovered
but will hopefully provide
better insight into the effects of
genetic variation in
rpoS
and the reasons for its persistence
within the natural
population.
| |
ACKNOWLEDGMENTS |
This work was supported by a BBSRC studentship.
We thank David Watts at Perkin-Elmer Applied Biosystems for assistance
in primer design, Tom Humphrey of PHLS in Exeter for providing some
strains for analysis, and K. Francis for the original rpoS primers.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of Food
Sciences, School of Biological Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, United
Kingdom. Phone: 01159515163. Fax: 01159516162. E-mail: christine.dodd{at}nottingham.ac.uk.
Professor Stewart died in February 1999, at age 47.
| |
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Applied and Environmental Microbiology, August 1999, p. 3582-3587, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
