Journal of Bacteriology, October 1999, p. 6449-6455, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Pathogenic Yersinia Species Carry a Novel,
Cold-Inducible Major Cold Shock Protein Tandem Gene Duplication
Producing both Bicistronic and Monocistronic mRNA
Klaus
Neuhaus,1
Kevin P.
Francis,1,2
Sonja
Rapposch,1
Angelika
Görg,3 and
Siegfried
Scherer1,*
Institut für Mikrobiologie,
FML-Weihenstephan,1 and Institut
für Allgemeine
Lebensmitteltechnologie,3 Technische
Universität München, 85350 Freising-Weihenstephan,
Germany, and Xenogen Corporation, Alameda, California
945012
Received 10 March 1999/Accepted 15 July 1999
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ABSTRACT |
Inverse PCR was used to amplify major cold shock protein (MCSP)
gene families from a diverse range of bacteria, including the
psychrotolerant Yersinia enterocolitica, which was found to have two almost identical MCSP coding regions (cspA1 and
cspA2) located approximately 300 bp apart. This tandem gene
duplication was also found in Y. pestis, Y. pseudotuberculosis, and Y. ruckeri but not in other
bacteria. Analysis of the transcriptional regulation of this MCSP gene
in Y. enterocolitica, performed by using both reverse
transcriptase-PCR and Northern blot assays, showed there to be two
cold-inducible mRNA templates arising from this locus: a monocistronic
template of approximately 450 bp (cspA1) and a bicistronic
template of approximately 900 bp (cspA1/A2). The former may
be due to a secondary structure between cspA1 and
cspA2 causing either 3' degradation protection of
cspA1 or, more probably, partial termination after
cspA1. Primer extension experiments identified a putative
transcriptional start site (+1) which is flanked by a cold-box motif
and promoter elements (
10 and 35) similar to those found in
Escherichia coli cold-inducible MCSP genes. At 30°C, the
level of both mRNA molecules was negligible; however, upon a
temperature downshift to 10°C, transcription of the bicistronic mRNA
was both substantial (300-fold increase) and immediate, with transcription of the monocistronic mRNA being approximately 10-fold less (30-fold increase) and significantly slower. The ratio of bicistronic to monocistronic mRNA changed with time after cold shock
and was higher when cells were shocked to a lower temperature. High-resolution, two-dimensional protein gel electrophoresis showed that synthesis of the corresponding proteins, both CspA1 and CspA2, was
apparent after only 10 min of cold shock from 30°C to 10°C. The
data demonstrate an extraordinary capacity of the psychrotolerant Y. enterocolitica to produce major cold shock proteins upon
cold shock.
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INTRODUCTION |
The genus Yersinia
contains 11 species of gram-negative facultative rods, including three
pathogens of humans, Yersinia enterocolitica, Yersinia
pseudotuberculosis, and Yersinia pestis, and the
salmonid fish pathogen, Yersinia ruckeri (20).
Y. enterocolitica causes gastroenteritis in humans and
animals through its ingestion in contaminated food or water
(34). It is the ability of Y. enterocolitica to
grow at temperatures as low as 5°C (2, 35) that has
mainly resulted in cases of bacterial septicemia from the transfusion of stored refrigerated blood products (31). On the other
hand, Y. enterocolitica can grow at temperatures as high as
42°C (34).
The molecular basis of cold tolerance and the roles played by
cold-inducible proteins are still poorly understood. The major focus of
recent work has been on mesophilic bacteria such as Escherichia coli and Bacillus subtilis (reviewed in references
15 and 42). Initial work showed
that when an exponential-phase culture of E. coli is shifted
from 37 to 10°C, a novel set of at least 13 proteins is induced
(24). Identification of a number of these cold shock
proteins has revealed several polypeptides that are involved in
transcription and translation. This has led to the suggestion that the
cold shock response is an adaptive mechanism facilitating protein
synthesis at low temperature (25) and, as reported recently,
at early exponential-phase growth (4). In contrast to the
relatively minor level of induction (2- to 10-fold) observed for most
cold shock proteins during a temperature downshift in E. coli, the induction of a novel protein (initially designated as
F10.6) was found to be considerably higher (24). This
70-amino-acid polypeptide was shown to be induced 200-fold following a
shift from 37 to 10°C (24) and was subsequently termed the
major cold shock protein (MCSP) or CspA (10). MCSPs are
extremely widespread in eubacteria (reference 8 and
this study) and belong to the most conserved group of nucleic
acid-binding proteins yet defined in nature: the cold shock domain
(CSD) proteins (40). They are characterized by the ability
to preferentially bind to single-stranded nucleic acid sequences
containing an ATTGG/CCAAT motif (16, 28, 36, 37,
40). In both prokaryotic and eukaryotic organisms, this ability
has been shown to be due to two RNA-binding motifs, RNP-1 (KGFGF) and a
partial RNP-2 (VFVH) (14, 40). Although a number of roles
have been proposed for MCSPs, their most likely function is as
molecular chaperones involved with the unfolding of mRNA secondary
structures formed at low temperature (17, 22, 23).
E. coli, B. subtilis, Bacillus cereus,
and Pseudomonas fragi have all been shown to have families
of MCSP homologues, ranging from three in B. subtilis to
nine in E. coli (16, 28, 29, 42). These proteins
are all around 70 amino acids. Comparison of the four cold-inducible
MCSPs of E. coli, CspA, CspB, CspG, and CspI, shows these
proteins to have considerable homology (30, 39, 42).
Moreover, alignment of the 5' untranslated mRNA pertaining to these
four MCSPs shows this homology to extend beyond each coding region,
with the leading 160 bp of cspB and cspG having 70% or greater identity (30, 39). These four MCSPs of
E. coli are differentially regulated at low temperature
(7, 39).
To our knowledge, no information on the cold shock response of the
psychrotolerant bacterium Y. enterocolitica is available. In
this study, we show that Y. enterocolitica has an MCSP gene duplication that is induced by cold shock to give both monocistronic and bicistronic mRNAs. Furthermore, we demonstrate that both MCSPs are
translated and that transcription and translation upon cold shock are
extremely rapid.
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MATERIALS AND METHODS |
Inverse PCR amplification of multiple MCSP gene sequences from
gram-positive and gram-negative bacteria.
Genomic DNA was purified
(1) from a wide range of both gram-positive and
gram-negative bacteria, including the psychrotolerant Bacillus
weihenstephanensis (WSBC10201) and the mesophilic B. cereus (WSBC10028), Enterococcus faecalis (NCTC 775),
E. coli (W3110), Klebsiella pneumoniae (NCTC
9633), Listeria monocytogenes (ATCC 23074),
Micrococcus luteus (NCTC 2665), Proteus vulgaris (NCTC 4175), Pseudomonas aeruginosa (PAO1), Salmonella
typhimurium (LT2), Staphylococcus aureus (RN4220), and
Y. enterocolitica (NCTC 10460). Approximately 1 µg of each
DNA was dissolved in 50 µl of water, which was then subdivided into
six 8-µl aliquots. Five aliquots were cut individually with the
restriction enzymes AluI, HhaI, HpaII,
MboI, and RsaI (Roche Diagnostics GmbH, Mannheim, Germany), while the sixth aliquot of uncut DNA acted as a control. After heat inactivation of the restriction enzyme, the cut DNAs were
self-ligated (1 U of T4 DNA ligase; Promega) for 4 h at 16°C.
Using a compilation of MCSP DNA sequences gained from more than 30 species of bacteria (8), three degenerate oligonucleotide primers were designed (two pairs) with which to perform inverse PCR.
The sequences of these primers are as follows: CSPIF1, 5' [AG][AG]I
GA[CT] GTI TTC GT[AT] CA[CT] TT[CT] I[GC]I GC 3'; CSPIF2, 5'
GGI T[AT]C AAA [AT]CI [CT]T[AG] CAI GAA GG[CT] CA 3'; and
CSPIR1, 5' [GC][GC][AT] [GT]I[AG] AT[AG] AAI CC[AG] AAI
CCT TTI TC 3' (bracketed nucleotides show the degeneracies used, and I
represents inosine). PCR was performed with a Techne Progene automated
thermocycler with 0.2-ml thin-walled PCR tubes. Reactions were carried
out in 50-µl volumes containing 5 µl of 10× PCR buffer (supplied
with Taq DNA polymerase; Eurogentec), 2 mM
MgCl2, 100 pmol of each oligonucleotide primer, 0.2 mM each
deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), 1 U of
Taq DNA polymerase (Eurogentec), and 1 µl of a ligation
mixture or uncut DNA. PCR conditions were as follows: 2 min at 95°C,
followed by 35 cycles at 95°C for 15 s, 50°C for 2 min, and
72°C for 3 min, with a final extension at 72°C for 5 min. Amplified
products were analyzed on a 1.5% agarose gel (NuSieve; FMC
BioProducts). Reactions containing PCR products were then run on a
low-melting-point agarose gel (SeaPlaque GTG; FMC BioProducts), and DNA
fragments were liquid nitrogen band extracted by using a freeze-thaw
procedure (32). Direct sequencing of PCR products was
performed with an ABI 373A sequencer (Perkin-Elmer Applied Biosystems)
with CSPIR1 and either CSPIF1 or CSPIF2 (depending on which
oligonucleotide was used to perform PCR) as sequencing primers. DNA
sequences gained from the latter procedure were then used to design
specific PCR primers to amplify complete MCSP genes, including their
missing central regions.
Preparation of RNA from Y. enterocolitica.
A 350-ml
culture of Y. enterocolitica was grown at 30°C to an
optical density at 600 nm of 0.5. This was then cold shocked to 10°C
in an ice bath. Ten-milliliter samples were taken before (control) and
shortly after cold shock (2 min) and then at 10, 20, 30, 45, 60, 90, and 120 min after shock. The cells were centrifuged, and the pellet was
frozen in liquid N2. Total RNA was isolated from the
pellets with guanidine-phenol buffer as described before (18).
Northern blot analysis of cold-induced Y. enterocolitica MCSP mRNA.
Northern blotting, with 20 µg of
total RNA, was carried out as described previously (27) with
the following changes. The RNA was blotted with a vacuum blotter at 70 mbar for 1 h. The membrane was then air dried at 37°C for 20 min
before it was cross-linked with 0.3 J/cm2. Immediately
after cross-linking, the membrane was prehybridized. The blot was
washed at 30°C, if not indicated otherwise, as follows: two times for
5 min each in 2× SSPE (1× SSPE is 0.15 M NaCl, 100 mM
NaH2PO4, and 1 mM EDTA [pH 7.7]), 20 min at
55°C in 0.2× SSPE, 20 min in blocking buffer, followed by 1 h
of conjugation with anti-digoxigenin (DIG) AP (Roche Diagnostics), two
times for 5 min each in blocking buffer, two times for 5 min each in
1× phosphate-buffered saline (plus 0.5% sodium dodecyl sulfate), two
times for 5 min each in assay buffer, and finally transferred to the
substrate solution (250 µM CDP-Star; Tropix) for 5 min at room
temperature. The blot was exposed to Curix HC1.000G film (Agfa Gevart,
Köln, Germany) for 3 to 15 min. Hybridization solution was made
with DIG-labelled oligonucleotide (YeA1-DIG, 5' GCC ACA ATA CTG TTT TGC
CAC AAT ATG T 3') or DIG-labelled PCR products amplified with the
primers Melmack (5' GCT GCT GGC ACG TAG TTA 3') and Alex (5' ACT GGG
ACT GAG ACC GG 3') in accordance with the Boehringer Mannheim manual.
Primer extension.
Primer extension was conducted as
described previously (26) with the following minor changes:
5 µg of total RNA (shock at 10°C for 10 min) was incubated with 4 µl of the primer YeA1R2(+1) (2 pmol; 5' GCC ACA ATA CTG TTT TGC CAC
3') and 2.6 µl of H2O for 1 min at 94°C and then 10 min
at 50°C. All subsequent steps were conducted at 50°C. A sequencing
reaction for a ladder was carried out with different plasmids and
primers in accordance with the manual of the Sequenase V2.0 DNA
sequencing kit (Amersham Pharmacia Biotech, Freiburg, Germany).
RT-PCR of cold-induced Y. enterocolitica bicistronic
cspA1/A2 mRNA.
Reverse transcriptase (RT)-PCR was
carried out with the primer YeRTR (5' GGC TAT CAC CTT CAT CGC 3') for
MCSP mRNA as described for primer extension but without using labelled
dATP. PCR was conducted by using the primers YeRTF (5' CAT CGG TTT GGA
CAC CAG AC 3') and YeRTR, with the following parameters: 95°C for
10 s, 50°C for 15 s, and 72°C for 20 s for 20 cycles.
Southern blot analysis of Y. enterocolitica DNA.
Ten micrograms of DNA was cut with the restriction enzymes
SspI, EcoRV, EcoRI. Fragments were run
on a 1.2% Tris-acetate-EDTA agarose gel and blotted on a nylon
membrane (Hybond-N+; Amersham Pharmacia Biotech).
Hybridization and washing were carried out as described for Northern
blotting above.
Prediction of secondary mRNA structure.
The secondary
structure of the bicistronic cspA1/A2 mRNA from Y. enterocolitica was obtained by using the folding software program
MFOLD (38, 43, 43a).
Two-dimensional polyacrylamide gel electrophoresis.
For the
preparation of protein samples, shocked cells were centrifuged and the
pellets were resuspended in solubilization buffer as described
previously (12). Cell lysis was performed by a single
passage through a French press (SLM Aminco Inc., Rochester, N.Y.) cell
at 20,000 lb/in2. The cell extract was then centrifuged for
45 min (15,400 × g at 4°C). Two-dimensional gel
electrophoresis of the supernatant was performed as described before
(11-13a) by using immobilized pH gradient (IPG) recipes (pH 4 to 7 and
pH 5 to 6) described previously (33). For analytical
purposes, samples of approximately 70 µg, for microseparation ca. 700 µg, of protein per gel were used. Proteins were resolved by
isoelectric focusing with Pharmacia's DryStrip kit. The protein
solution was applied at the anodic side of the IPG gel strips. The
sample was run into the gel at low voltages (gradient pH 4 to 7, 150, 300, and 600 V, each for 3 h; gradient pH 5 to 6, 150, 300, and
600 V, each for 5 h). Isoelectric focusing was done for 12 h
at 3,500 V (gradient pH 4 to 7) and for 12 h at 1,500 V and
12 h at 3,500 V (gradient pH 5 to 6). The IPG gel strips either
were used immediately for the second-dimension run or were stored at
80°C. The IPG gel strips were equilibrated two times as described
previously (12). In the second dimension, self-casted sodium
dodecyl sulfate-pore gradient gels on plastic backing (12)
with a gel size of 190 by 250 by 0.5 mm3 were run for
0.75 h at 300 V and 4.5 h at 600 V on a Multiphor electrophoresis unit (Pharmacia). The gels were stained with silver (3), or the proteins were blotted onto polyvinylidene
difluoride membranes (Millipore) with Tris-borate transfer buffer
(12) and stained with Coomassie brilliant blue R-250
(Serva). Selected protein spots either were directly applied to an
automated protein sequencer to obtain N-terminal amino acid sequences
or were digested with trypsin and the mass of the peptide fragments was
analyzed with an automated mass spectrometer (matrix-assisted laser
desorption ionization [MALDI]; Toplab, Martinsried, Germany).
Nucleotide sequence accession numbers.
Complete sequences of
MCSPs were deposited in the GenBank database and include E. coli
cspB-cspF (accession no. AF003590) and cspH (accession
no. AF003591), M. luteus cspA (accession no. AF019905),
P. aeruginosa cspA (accession no. U82822), S. aureus
cspB and cspC (accession no. AF003592 and AF003593), S. typhimurium cspH (accession no. AF006035), and Y. enterocolitica cspA1/A2 (accession no. U82821).
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RESULTS |
A cspA gene duplication in Y. enterocolitica.
Inverse PCR of bacterial genomic DNA, performed
with the degenerate MCSP oligonucleotide pairs CSPIR1-CSPIF1 and
CSPIR1-CSPIF2, resulted in the amplification of at least one MCSP
sequence from all bacteria tested (see Materials and Methods for the
list of strains). In the majority of cases, control PCRs containing
uncut genomic DNA did not give amplified products. Exceptions to this were E. coli and Y. enterocolitica, which were
found to give products of between 400 and 500 bp. Sequencing of the PCR
products from E. coli and Y. enterocolitica
confirmed these DNAs to each contain two MCSP sequences, E. coli having divergent MCSP coding regions (corresponding to
cspB-cspF and cspG-cspH, respectively) and
Y. enterocolitica having a tandem repeat (Fig.
1).
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FIG. 1.
Nucleotide sequence of cspA1/cspA2 from
Y. enterocolitica. cspA1 (upper protein sequence) and
cspA2 (lower protein sequence) differ only in the 13th and
15th amino acids and in the third amino acid of the C terminus.
Putative transcriptional start site (+1), putative promoter regions
(10, 35), and the cold-box are indicated. Sequence in boldface type
shows the position of the A1 probe (YeA1-Dig). The underlined sequence
indicated by upper facing arrows shows the putative transcriptional
termination structure of the monocistronic mRNA. The underlined
sequence indicated by lower facing arrows shows the transcriptional
termination structure of the polycistronic mRNA. The sequence
underlined by a wavy line in cspA1 can fold in an
antiparallel direction to the same region of cspA2, forming
an extensive secondary structure. EcoRV restriction sites,
which cut at positions +73, +579, and +909, are indicated by italics.
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Comparison of the two MCSP coding regions from Y. enterocolitica, designated cspA1 and cspA2,
shows these sequences to be almost identical (96.7%), with their
deduced protein sequences only differing in amino acids 13 and 15 (CspA1, DAG; CspA2, NAD) and a single amino acid at their C termini
(CspA1, VVAL; CspA2, VIAL). Furthermore, there is a large degree of
homology between the 5' untranslated regions of these two genes, with
the first 175 bp upstream of cspA1 and the corresponding 154 bp upstream of cspA2 showing approximately 70% identity.
Primer extension experiments identified the 5' end of the
cspA1/A2 mRNA as lying 197 bp upstream of the first coding
region (cspA1, ATG) (data not shown). This is probably the
transcriptional start site +1 because it is in the same position as
that in E. coli and is flanked by a cold-box motif and
promoter elements (10 and 35) similar to those found in E. coli MCSP genes (7, 10, 21).
Interestingly, the coding regions of the Y. enterocolitica
cspA1/A2 mRNA can fold and hybridize to each other, resulting in an extensive secondary structure. This secondary structure begins with
the 17th codon in cspA1 (GGU), which can bind in an
antisense direction to the 44th codon of cspA2 (ACC) and
vice versa (Fig. 1).
Transcription of Y. enterocolitica MCSP gene
duplication.
Northern blot analysis of Y. enterocolitica mRNA was conducted at both 30°C and after cold
shock to 10°C, hybridizing with the oligonucleotide A1 probe specific
to the 5' untranslated region of cspA1. This analysis showed
that two cold-inducible mRNA templates are produced from this MCSP
sequence (Fig. 2): a monocistronic template of approximately 450 bp (cspA1) and a bicistronic
template of approximately 900 bp (cspA1/A2). At 30°C, the
level of the bicistronic mRNA was low, with the level of monocistronic
mRNA almost negligible. However, upon a temperature downshift to
10°C, transcription was both substantial and immediate.
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FIG. 2.
Northern blots of Y. enterocolitica cold
shocked to 10°C. The signals were gained by a probe binding to the 5'
untranslated region of cspA1 (YeA1-Dig). Time after cold
shock is given in minutes. The upper signal is approximately 900 bp and
represents the bicistronic mRNA. The lower signal is approximately 450 bp and represents the monocistronic mRNA. CT, control.
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To confirm that the bicistronic transcript contained the complete
coding regions of both cspA1 and cspA2, RT-PCR
was performed with mRNA taken at 30°C and at 10°C after 30 min by
using an oligonucleotide back primer downstream of the cspA2
coding region. After 20 PCR cycles, an intense product of approximately
900 bp was obtained from the cold-shocked sample, with virtually no
product obtained from the control sample (data not shown). The former
product was sequenced and contained both cspA1 and
cspA2 gene sequences (Fig. 1).
To ensure that the A1 probe used in the Northern blots specifically
hybridized to cspA1/A2, a Southern blot analysis of Y. enterocolitica genomic DNA was conducted. This procedure confirmed that the A1 probe hybridized to only one region of this bacterium's DNA (data not shown). Furthermore, by including an EcoRV
digest of this DNA (a restriction enzyme known to cut before
cspA1, between cspA1/A2 and after
cspA2 [see Fig. 1], to give 456-bp and 330-bp DNA
fragments, respectively), it can be confirmed that both signals visible
on the Northern blots are mRNAs flanked by cspA1 and not a
second MCSP gene sequence.
Comparison of mono- and bicistronic mRNA synthesis.
To compare
the levels of monocistronic and bicistronic mRNA transcribed from this
MCSP gene sequence before and after cold shock, quantitative Northern
blot analysis was performed. mRNA was isolated at 30°C and compared
to a dilution range of mRNA taken at 10°C after 30 min. This showed
that at this time point the levels of bicistronic and monocistronic
mRNA were induced 300- and 30-fold, respectively (Fig.
3). However, at 30°C the level of
monocistronic mRNA was approximately fourfold higher than that of the
bicistronic mRNA. When cold shocked to 10°C, a high level of
bicistronic mRNA was observed at 30 min, whereas a high level of
monocistronic mRNA was not recorded until 60 min, after which time both
transcripts diminished. Neither transcript was visible 120 min after
the initial temperature downshift (Fig. 2).
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FIG. 3.
Northern blot of a dilution series of cold-shocked
Y. enterocolitica mRNA (10°C, 30 min) detected by using
the A1-probe. The relative amounts of bi- and monocistronic mRNA from
cspA1/A2 were determined with the software Image Master 1D
Elite (Amersham Pharmacia Biotech). The bicistronic mRNA shows a
300-fold increase; the monocistronic one shows a 30-fold increase.
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Comparison of the two mRNA transcripts shows that the ratio of
bicistronic and monocistronic mRNA increases with decreasing temperature (Fig. 4). At 0 and 5°C,
bicistronic mRNA is present at a high level, with relatively little
monocistronic mRNA appearing. In contrast, at 15°C, similar levels of
bi- and monocistronic mRNA are present, while at 20°C, the level of
monocistronic mRNA seems to be higher.
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FIG. 4.
Northern blots of cold-shocked Y. enterocolitica mRNA obtained at different temperatures with the A1
probe. Each cold shock lasted for 20 min. Changes in the proportion of
bicistronic and monocistronic mRNA are most obvious at 15 and 20°C.
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Cold-induced synthesis of Y. enterocolitica CspA1 and
CspA2.
Protein extracts gained from cultures of Y. enterocolitica that were grown at 30°C and then cold shocked at
10°C for increasing periods of time were analyzed by two-dimensional
gel electrophoresis. Initial studies using conventional gel gradients
between pH 4 and 7 identified MCSPs around 7 kDa (Fig.
5A). However, it was not possible to
determine how many separate MCSPs this spot contained. To increase
resolution, protein extracts were focused in a narrow immobilized pH
gradient between pH 5 and 6, over 18 cm, which to our knowledge has not
been used before. As can be seen in Fig. 5B, the protein spot of
interest actually consists of at least three proteins (marked 1, 2, and
3 on Fig. 5B). The N-terminal sequences of each of these spots was
determined. Since it was unclear which of the spots 2 or 3 contains
CspA2 (or another unknown MCSP), a peptide mass fingerprint (MALDI) was
executed with each of these spots. The data are summarized in Table
1. The mixture of Csps in spot 2 is most
probably due to carryover.
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FIG. 5.
Two-dimensional gels from Y. enterocolitica.
Molecular weights and pH values are indicated. MCSPs are indicated by
downward arrows. (A) Windows of the broad-range gels showing proteins
from pH 4 to 9 at 30°C and after a shock of 60 min to 10°C; (B)
windows of the narrow-range gels from pH 5 to 6 of the control (30°C)
and shock experiments to 10°C for 10 min and 60 min.
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MCSP tandem gene duplications in other species of
Yersinia.
To determine whether the MCSP gene duplication
found in Y. enterocolitica was present in other species of
Yersinia, three additional members of this genera were
investigated. Y. pseudotuberculosis (biotype III;
environmental isolate from H. Wolf-Watz, University of Umea, Umea,
Sweden) and Y. ruckeri (NCIMB 1316) were both probed by PCR,
using the oligonucleotide primers CSPIR1 and CSPIF2, and in both cases
gave a 450-bp product (similar in size to that gained from Y. enterocolitica). Sequencing of these DNA fragments (data not
shown) confirmed the flanking regions of each product to be MCSP
sequences homologous to Y. enterocolitica cspA1/A2.
Analysis of the Y. pestis database (Pathogen Sequencing
Group, Sanger Centre, Cambridge, United Kingdom) (37a)
confirmed an MCSP gene duplication to also be present in this
Yersinia species. Due to a number of unknown bases being
present in the latter sequence, this entire region of DNA was PCR
amplified and resequenced (Y. pestis DNA was a gift from R. Titball, Defence Evaluation Research Agency, Porton Down, United
Kingdom). Like those of Y. enterocolitica, the sequences of
CspA1 and CspA2 of Y. pestis show only minor changes.
Furthermore, both of these proteins are 94% identical to their
comparative homologues in Y. enterocolitica. Alignment of
the DNA sequences of both of these MCSP gene duplications, Y. enterocolitica cspA1/A2 with Y. pestis cspA1/A2, shows
that there is considerable homology (85% identity) throughout these entire sequences (approximately 950 bp), including the 5' untranslated region (data not shown).
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DISCUSSION |
Only Yersinia species carry a tandem cspA
gene duplication.
Recently, it has been shown that
Lactococcus lactis has two sets of MCSP genes,
cspA-cspB and cspC-cspD, both being separated by
approximately 300 bp. Comparison of these two MCSP loci showed them to
be homologous, with 79% identity over 800 bp (41).
Similarly, in this study we amplified two MCSP loci from E. coli that are located at 35 and 22 min on the chromosome. Each of
these loci again contains two MCSP genes, cspB-cspF and
cspG-cspH, that are separated by approximately 300 bp and
show high levels of homology (more than 70% identity over 750 bp). We
found no evidence to suggest that S. typhimurium has the
cspBF-cspGH duplication found in E. coli,
although subsequent work has demonstrated these sequences to be present
in Shigella species (data not shown). The high levels of
homology shown between these genes (loci) give strong evidence that
they have arisen by sequence duplication.
In contrast to Y. enterocolitica, all of the above MCSP
genes of L. lactis and E. coli are transcribed
divergently and monocistronically. In each of the latter bacteria, it
is the single MCSP locus that has been duplicated and not tandem genes,
as in the case of Y. enterocolitica, Y. pestis,
Y. pseudotuberculosis, and Y. ruckeri. Furthermore, although families of MCSPs have been identified in a wide
range of bacteria (16, 17, 28, 29, 42), Y. enterocolitica is the first bacterium in which bicistronic MCSP
mRNA has been found. This finding is especially intriguing in this
particular bacterium due to its exceptional ability to grow at low temperature.
Differential appearance of mono- and bicistronic
cspA1/A2 mRNA.
It has been shown that cspA
mRNA is constitutively transcribed in E. coli but that, at
elevated temperatures, this mRNA is rapidly degraded and may not be
translated. Following cold shock, cspA mRNA is specifically
and temporally stabilized (9). These findings could help to
explain the immediate and substantial quantities of bicistronic mRNA
produced by Y. enterocolitica upon cold shock from 30 to
10°C (Fig. 2). A sequence that is characteristic of a conventional
transcription termination site can be found downstream of the
cspA2 coding region (Fig. 1). This site, which is
approximately 900 bp downstream of the +1, is most probably where the
bicistronic messenger terminates.
With shock at temperatures not quite as low, monocistronic mRNA is
produced predominantly (Fig. 4); this may be sufficient to overcome the
impediment caused by this mild cold shock. The most significant
difference between the appearance of these two transcripts at 10°C is
that bicistronic mRNA is immediately available for translation at
10°C, whereas monocistronic mRNA does not significantly emerge until
30 min later (Fig. 2).
The ratio of monocistronic to bicistronic mRNA increases with the
duration of the cold shock (Fig. 2) and decreasing shock temperature
(Fig. 4). This change of the ratio of monocistronic to bicistronic mRNA
could be reflective of transcription termination downstream of
cspA1. Although a classical termination structure does not
appear to be present between cspA1 and cspA2,
sequence homology which indicates the formation of a large loop
structure (Fig. 1) exists; this structure may act as a
temperature-dependent second termination site. It cannot be excluded,
on the other hand, that this loop structure acts as 3' degradation
protection. Investigations of the rpsO operon in E. coli showed that a secondary structure (t1) is located between
rspO and pnp, which can act either as a
terminator or as 3' degradation protection. A second terminator (t2) is
found downstream of pnp. Therefore, rpsO is
sometimes transcribed monocistronically and sometimes bicistronically
together with pnp (5, 19). Further investigations
of the cspA1/A2 tandem in Yersinia will show
whether a similar mechanism occurs in this bacterium.
Both CspA1 and CspA2 are synthesized.
In case the bicistronic
mRNA indeed contributes to an elevated cold shock response, it should
be fully translated; i.e., CspA2 should be present after cold shock. By
electrophoresing proteins on two-dimensional gels with an extremely
narrow pH range between 5 and 6 (Fig. 5B), it is possible to separate
cold-inducible MCSPs. At least three cold-inducible MCSPs could be
found, CspA1, CspA2, and CspB (see Results). Surprisingly, CspA1 and
CspA2 separate at the narrow-range two-dimensional gel, although their
sequences differ by only three amino acids (see Results). We conclude
that CspA2 is synthesized and contributes to the cold shock response of
Y. enterocolitica.
Does Y. enterocolitica have a higher translational
capacity for MCSP than E. coli?
Studies in E. coli have shown that cspA mRNA is maximally induced
30-fold following a temperature downshift from 37 to 15°C and that
this peak induction occurs after approximately 60 min at this lower
temperature (9). These data correspond well with the
induction of monocistronic cspA1 mRNA in Y. enterocolitica (Fig. 2). In contrast, a high level of bicistronic
cspA1/A2 mRNA in Y. enterocolitica occurs after
only 30 min and is induced at least 300-fold, 10-fold more than the
monocistronic transcript (Fig. 2 and Fig. 3) of Y. enterocolitica and E. coli. If it is true that the
basal levels of the monocistronic MCSP mRNAs are similar in both
bacteria at non-cold shock temperatures, Y. enterocolitica would have a much greater translational capacity than E. coli upon cold shock with respect to these single MCSP sequences (loci).
Additionally, the fact that this transcript contains two copies of the
coding region, which are both translated, means that Y. enterocolitica is able to synthesize this protein more effectively than bacteria that have monocistronic MCSP mRNAs only, such as E. coli, assuming that translation levels of these two mRNAs are comparable. Indeed, synthesis of CspA1 and CspA2 is clearly seen only
10 min after cold shock. However, in order to finally conclude that the
rapid accumulation of these MCSPs is essential for Y. enterocolitica's superior ability to adapt to low temperature, a
knockout mutant of cspA2 needs to be constructed.
| |
ACKNOWLEDGMENTS |
K. Neuhaus and K. P. Francis contributed equally to this work.
The majority of the gene sequences reported in this manuscript were
discovered by Kevin P. Francis in the laboratory of Gordon S. A. B. Stewart at the University of Nottingham (ROPA grant 42/CEL 04626). We thank Günther Boguth and Christian Obermaier for
support and technical assistance in preparing the two-dimensional gels.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Mikrobiologie, Forschungszentrum für Milch und
Lebensmittel Weihenstephan, Technische Universität München,
Weihenstephaner Berg 3, 85350 Freising, Germany. Phone: 49 8161 713516. Fax: 49 8161 714512. E-mail:
Siegfried.Scherer{at}lrz.tum.de.
| |
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Abstract
Introduction
