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Infection and Immunity, June 2001, p. 3972-3979, Vol. 69, No. 6
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.6.3972-3979.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Gene Fragments Distinguishing an
Epidemic-Associated Strain from a Virulent Prototype Strain of
Listeria monocytogenes Belong to a Distinct Functional
Subset of Genes and Partially Cross-Hybridize with Other
Listeria Species
Martin
Herd and
Christine
Kocks*
Institute for Genetics, University of
Cologne, D-50674 Cologne, Germany
Received 16 November 2000/Returned for modification 6 February
2001/Accepted 13 March 2001
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ABSTRACT |
Most major food-borne outbreaks of listeriosis in Europe and in the
United States have been caused by genetically closely related
Listeria monocytogenes strains of serotype 4b. In order to
assess whether genomic loci exist that could underlie this increased
epidemic potential, we subtracted the genome of the virulent prototype
L. monocytogenes strain EGD from a prototype epidemic
strain. A total of 39 DNA fragments corresponding to 20% of an
estimated total of 150 to 190 kb of differential genome material were
isolated. For 21 of these fragments, no function on the basis of
homology could be predicted. Of the remaining 18 fragments, 15 had
homologies to bacterial surface proteins, some of which have been
implicated in virulence mechanisms such as cell invasion, adhesion, or
immune escape. Southern hybridization of arrays containing the
epidemic-clone-specific DNA segments with genomic DNA of different
L. monocytogenes strains was consistent with the current
lineage division. Surprisingly, however, some of the fragments
hybridized in a mosaic-like fashion to genomes of two other
Listeria species, the animal pathogen L. ivanovii and the nonpathogen L. innocua. Taken
together, our results provide a starting point for the identification
of epidemic-trait-associated genes.
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INTRODUCTION |
The gram-positive, opportunistic
bacterium Listeria monocytogenes is responsible for severe
food-borne infections with a high case fatality rate in susceptible
animal and human hosts (16, 44). Human listeriosis occurs
as sporadic disease or in the form of outbreaks (epidemics) which can
affect up to several hundred people, in particular elderly, very young,
or immunocompromised individuals. Symptoms of primary infection range
from barely apparent to distinct flu-like symptoms and self-limiting
diarrhea. After an extended incubation period, susceptible individuals
may develop listeriosis, which may manifest itself as meningitis,
meningoencephalitis, or sepsis, or in the case of pregnancy as
abortion, miscarriage, or severe generalized infection of the newborn.
Of the 16 or so L. monocytogenes serotypes known, three
serotypes, 4b, 1/2b, and 1/2a, are responsible for most human cases (16, 44). Molecular and other typing methods divide the
species L. monocytogenes into distinct phylogenetic lineages
separating the serotypes 4b, 3b, and 1/2b from the serotypes 1/2a and
1/2c (6, 38, 42). Serotype 4b strains are responsible for
the majority of sporadic disease as well as most major outbreaks of food-borne listeriosis in Europe and North America since 1981. By
multilocus enzyme electrophoresis, a specific 4b strain, representing 1 of 82 electrophoretic types of L. monocytogenes, was
associated with epidemic disease outbreaks in California in 1985, Switzerland between 1983 and 1987, and France in 1992 (6, 26,
38). A 4b strain with a closely related electrophoretic type was
implicated in two outbreaks in Boston in 1979 and 1983 (6,
38).
L. monocytogenes possesses a number of well-characterized
virulence factors that play a role in its facultative intracellular lifestyle and its capacity to circumvent the humoral immune system by
spreading from cell to cell within tissues (11, 45).
Although no correlation between epidemic prevalence and increased
virulence in a number of animal models and cell culture tests has been
found to date for L. monocytogenes 4b strains (28,
46), one should nevertheless consider the possibility of genetic
loci conferring additional pathogenic traits (12) to the
strains prevalent in epidemics. These traits may include properties
(not measurable by existing virulence tests) that allow the adaptation
to specific environmental conditions and that can influence the course
or the outcome of an infection.
One way to identify genetic loci encoding such traits is to employ
bacterial genome subtraction (1, 9, 36). Despite the
apparent genetic distinctness of L. monocytogenes serotype 4b strains, only three genes have been identified to date which are
characteristic of serotype 4b strains. These genes were isolated on the
basis of their involvement in expression of cell wall teichoic acid-associated, serotype 4b-specific surface antigens (30, 40).
In order to assess whether genetic loci exist that distinguish epidemic
L. monocytogenes strains, we subtracted the genome of the
virulent, experimentally best-characterized strain (EGD serotype 1/2a,
whose completed genome sequence is pending publication) from a
prototype epidemic strain (F.4565, serotype 4b) that had caused 142 cases of listeriosis including 48 deaths during an outbreak in Los
Angeles in 1985 (31). We found that about 5 to 6% of the
genome of strain F.4565 does not hybridize to strain EGD DNA. We have
isolated 39 such fragments, 35 of which were absent and 4 of which were
strongly divergent from the genome of strain EGD. A large portion of
these DNA regions code for bacterial surface determinants and hence are
likely to play a role in the interaction with the environment or the
host organism. We have devised a screening method to test the
occurrence of these fragments in the genomes of other
Listeria species or other L. monocytogenes strains, thereby opening the way to search for
epidemic-trait-associated genes.
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MATERIALS AND METHODS |
Bacterial strains.
L. monocytogenes strains
F.4565 (bacterial collection no. [BAC] 239), G.3129 (BAC 266), F.7441
(BAC 262), L.4486a (BAC 240), L.2190a (BAC 265), L.2192 (BAC 260),
F.1092 (BAC 267), F.8353 (BAC 259), G.0039 (BAC 258), L.5089 (BAC 261),
F.7390 (BAC 264), and L.735 (BAC 263) (7; see also Table 2) were
provided by J. McLauchlin, Food Hygiene Laboratory, Central Public
Laboratory, London, England. L. monocytogenes strain LO28
(BAC 089), L. innocua Rham
(BAC 042)
CLIP11262T, L. innocua (BAC 269) CLIP12511 (type strain), L. ivanovii (BAC 085) CLIP12510, L. grayi (BAC
273) ATCC 25401, and B. subtilis 168 trpC2 (BAC
274) were obtained from P. Cossart, Institut Pasteur, Paris, France.
L. monocytogenes strain EGD (BAC 146) was obtained from W. Goebel, Theodor-Boveri-Institut, Würzburg, Germany. The
Escherichia coli strain used was JM101 (BAC 098). L. monocytogenes L028 
actA hly (BAC 200)
is an isogenic mutant of strain L028 carrying deletions of
actA and hly (C. Kocks, unpublished data).
Purification of genomic DNA, Southern blotting, and "reverse"
Southern blotting.
Molecular biology methods were carried out
using standard procedures (5) unless indicated otherwise.
High-molecular-weight DNA was prepared from saturated liquid bacterial
culture, taking care to remove all RNA. In order to verify the absence
of plasmids, an alkaline lysis type plasmid DNA preparation was carried
out with strains F.4565, L.4486a, L. innocua BAC 042, and
L. ivanovii BAC 085 and the equivalent of up to 2 ml of
satured liquid culture loaded onto an agarose gel. For Southern
blotting, 200 ng of RsaI-digested genomic DNA was separated
and blotted onto positively charged nylon membranes (Hybond N+;
Amersham Pharmacia) by downward capillary transfer (5).
Membranes were hybridized in Puregene Hyb-9 DNA hybridization solution
(Gentra Systems) to 10 ng of PCR product labeled with
[
-32P]ATP (Megaprime kit; Amersham Pharmacia). Washing
steps were carried out under low-stringency conditions (2× SSC [1×
SSC is 0.15 M NaCl plus 0.015 M sodium citrate] at room temperature), and the membrane was exposed to X-ray film. For reverse Southern blotting, plasmid inserts were amplified by PCR using adapter-specific oligonucleotides. Approximately 20 ng of plasmid DNA or PCR products were spotted onto nylon membranes either by hand or by using a VP408
Multi-Blot replicator (V&P Scientific, Inc.) and hybridized to 25 ng of
radioactively labeled, RsaI-digested genomic DNA.
Establishment of genomic subtraction conditions suitable for
Listeria genomes.
HaeIII-digested 
X174
replicative-form (RF) DNA (5,389 bp) was added to L. monocytogenes EGD DNA (3,150 kb) at a molar ratio of 1:1 and
subtracted as described below. Assuming a similar fragment length
distribution, the initial ratio of phage-specific fragments to other
DNA fragments was 1/589. We cloned and sequenced the PCR-amplified
material from this subtraction as described below. Four out of 18 randomly selected clones were identified as tester-specific X174 RF
HaeIII fragments, corresponding to a ratio of 1/4.5. Thus,
enrichment for tester-specific fragments was 130-fold. A similar
enrichment factor (132-fold) was found in a second control subtraction
in which DNA of L. monocytogenes strain L028 was used as
tester and DNA of an isogenic mutant, LO28 actA
hly lacking genes actA and hly, was
used as driver. In this case, the subtraction efficiency was assessed
for an actA fragment by PCR as described below for
plcA, iap, and hly.
Genome subtraction and plasmid library construction.
Genome
subtraction was performed with the PCR-Select Bacterial Genome
Subtraction kit (Clontech) according to the manufacturer's instructions except for the hybridization temperature. Genomic DNA was
digested with RsaI and subtractive hybridization was carried out at 60°C, a temperature adapted to the low GC content of the Listeria genome (17). Tester-specific fragments
were amplified by two rounds of suppression PCR that yielded a mixture
of fragments corresponding in size to RsaI-restricted
genomic DNA. The subtraction efficiency was tested with PCR by
comparing the concentration of RsaI fragments of three known
genes, plcA (KO79/80
5'-TCGCGTTACCTGGCAAATAGATGG-3'/5'-TCAAATCATCGACGGCAACCTCGG-3'), hly (KO83/84
5'-CCGAACTGCATGCCGAATTTGC-3'/5'-CAATCTCAACATTTACCATGGG-3'), and iap (KO77/78
5'-AAAGCGGTGACACTATTTGGGC-3'/5'-TTGTGTTTGTAGATGGTGCAGG-3'), in equal amounts of PCR-amplified subtracted and unsubtracted material. The PCR products enriched for tester-specific fragments were
ligated into plasmid vector pGEM-T Easy (system I; Promega) and
transformed into competent E. coli cells. Plasmid DNA of 355 clones from the subtracted library and of 125 clones from the unsubtracted library was prepared with the QIAprep 8 Miniprep kit
(Qiagen) and analyzed by restriction with EcoRI to test for the presence of inserts and to normalize plasmid DNA concentration. Insert sizes ranged from approximately 0.2 to 2 kb with an average length of 800 bp (consistent with the size distribution of
RsaI fragments of genomic L. monocytogenes DNA).
Sequence analysis and database searches.
Cycle sequencing
was carried out using the BigDye Termination kit (PE Applied
Biosystems) and an ABI Prism 377 DNA sequencer. Inserts were sequenced
by one pass in each direction using vector-specific oligonucleotides.
Homology searches at the nucleotide level (all nonredundant GenBank
plus EMBL plus DDBJ plus PDB sequences) or at the amino acid level (all
nonredundant GenBank CDS translations plus PDB plus SwissProt plus PIR
plus PRF sequences) were carried out at the National Center for
Biotechnology Information using BLASTN 2.0.12 and BLASTX 2.1.1 (4). Expect values lower than 0.05 were considered to be
potentially significant. If necessary, internal fragment-specific
oligonucleotides were used to obtain the complete sequence of fragments
with homology to known genes. Fragments with homologies to unknown
genes or no homology to database entries were sequenced to about 300 to
400 bp from both ends.
Nucleotide sequence accession numbers.
The sequences
described in Table 1 have been deposited in the EMBL database and
assigned accession numbers AJ410352 to AJ410411. They can also be
accessed at http://www.uni-koeln.de/~aeg14/Kocks/index.html.
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RESULTS |
Five to 6% of the L. monocytogenes strain F.4565
genome did not hybridize to the genome of L. monocytogenes
strain EGD.
Genomic subtractions were carried out using a
PCR-based subtractive hybridization method (1). Effective
subtraction conditions for L. monocytogenes were established
as detailed above in Materials and Methods. In order to identify DNA
fragments specific for an epidemic clone of L. monocytogenes
(strain F.4565; serotype 4b) (31) that belongs to a
genetically closely related subgroup of strains that has caused major
outbreaks of listeriosis in America and Europe (6, 26,
38), we subtracted the virulent prototype L. monocytogenes strain EGD (serotype 1/2a) from this epidemic strain. Subtraction efficiency for the L. monocytogenes
genes plcA, hly, and iap (all shared
by both strains) ranged from 64-fold for plcA to 8-fold for
hly to no detectable subtraction for iap (data
not shown).
Plasmids from libraries of PCR-amplified subtracted and unsubtracted
material were spotted onto positively charged nylon membranes and
hybridized with radioactively labeled whole-genome probes of tester
(L. monocytogenes strain F.4565) and driver (L. monocytogenes strain EGD) DNA under low-stringency conditions. A
total of 45 of 270 clones from the subtracted library versus 5 of 90 clones from the unsubtracted library did not hybridize with driver DNA, indicating threefold enrichment. Since the differentially hybridizing fragments from the nonsubtracted library were in the average size range
(see Materials and Methods), one can conclude from the frequency of
these clones (5 out of 90) that in the order of 5 to 6% of the genome
of the epidemic L. monocytogenes clone strain F.4565 does
not hybridize with the genome of L. monocytogenes strain EGD.
A total of 50 differentially hybridizing clones were isolated and
sequenced. Seven of these turned out to be isolated more
than once. The
inserts of the 41 remaining, unique clones were
amplified by PCR,
arrayed onto positively charged nylon membrane,
and reanalyzed. Figure
1A shows that 39 clones hybridized
differentially
with the genome of the tester strain
L. monocytogenes F.4565.
To verify these results, 6 of the 39 fragments were tested by
Southern blotting under low-stringency
conditions (data not shown).
All six fragments hybridized specifically
to the tester strain
(
L. monocytogenes strain F.4565) and to
another epidemic strain
of serotype 4b (
L. monocytogenes
strain L.4486a from the Swiss
outbreak in 1983), while none of the
fragments showed hybridization
to the driver strain (
L. monocytogenes EGD, serotype 1/2a) or
to
L. monocytogenes strain L028 (serotype 1/2c). Two of the six
fragments cross-hybridized to different extents with
L. innocua DNA (U46 and 143; see also Fig.
1 and below).
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FIG. 1.
Identification of 39 gene fragments specific for the
epidemic L. monocytogenes strain F.4565 and
cross-hybridization with other Listeria species, with
reverse Southern blots of replica DNA arrays. PCR products were spotted
four times each at 20 ng per spot onto positively charged nylon
membranes and hybridized to whole-genome probes of Listeria
strains. Membranes were washed under low-stringency conditions. The
first row of the array contained eight known DNA fragments as
hybridization controls. The second row contained two sets of four
unrelated DNA sequences at 80, 40, 20, and 10 ng per spot as negative
controls. The third row contained four sets with no DNA followed by
three fragments common to the tester and driver strains (no. 64, 60, and 62). Epidemic-clone-specific fragments were spotted from left to
right, starting with no. 17B and ending with no. 16 containing the
270-bp flanking vector sequence. Fragments 153 and 121 gave no signal
and were excluded from the analysis. 65B is the same as U46. (A) A
total of 39 fragments hybridized differentially with tester DNA. (B)
Nine L. monocytogenes strain F.4565-specific fragments gave
strong signals with L. innocua DNA, five gave signals with
L. ivanovii DNA, and none gave signals with L. grayi DNA.
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The absence of the 39 differentially hybridizing fragments from the EGD
genome were confirmed by BLASTN and FASTA nucleotide
similarity
searches against the completed genome sequence of
L. monocytogenes strain EGD-e (which corresponds to our driver
strain).
Only 4 of the 39 fragments showed significant homologies to
EGD-e
genome sequences, mainly in coding regions (European Consortium,
personal communication). Two of these fragments (125 and 015)
had at
one end a short sequence region (of about one-sixth of
their respective
lengths) that had high homology to EGD-e sequences,
indicating a
junction between conserved and nonconserved F.4565
genome regions. Four
differentially hybridizing fragments could
be detected with FASTA (162, 60% identity in 394 nucleotides [nt];
73, 66% identity in 395 nt;
128B, 69% identity in 341 nt; and
157, 71% identity in 416 nt
[European Consortium, personal communication]).
(Fragment number 157 showed a weak, barely detectable hybridization
signal representing our
detection limit.) Thus, in these cases,
homologous sequences were
present in the EGD-e genome but showed
a high level of sequence
diversity. In contrast to these findings,
13 nondifferential control
fragments showed strong homologies
to EGD-e sequences with high
conservation ranging from 88 to 98%
similarity (European Consortium,
personal
communication).
Many strain F.4565-specific gene fragments had homologies to
surface proteins involved in virulence.
The results of amino acid
homology searches in translated public nucleotide or protein databases
with the 39 translated L. monocytogenes strain
F.4565-specific genome fragments are summarized in Table
1. A total of 21 fragments showed no
significant homology to database entries ("no hit") or were
homologous to proteins of unknown function. Eleven fragments showed
homologies to surface proteins of L. monocytogenes or to
other gram-positive bacteria, including one to a leucine-rich protein
interaction motif of a plant disease resistance gene, three to ABC
transporters (fragments 29, 120, and 125), one to a locus involved in
surface antigen expression, one to a component of a type II restriction
system, and one to a metabolic enzyme. In contrast to these findings, when we analyzed 13 randomly selected nondifferential clones (i.e., hybridizing to both the tester and driver L. monocytogenes
strains) we found that only one was a "no hit" and only two had
homology to bacterial surface components, whereas 10 had homology to
various enzymes. The distribution of database hits in different protein categories is graphically displayed in Fig.
2. A total of 41% of the differential
fragments showed homology to bacterial surface components. Many of
these have previously been implicated in virulence mechanisms, such as
L. monocytogenes internalins (host cell recognition and
invasion) (11, 19), the alpha C protein of group B
streptococci (repeats involved in immune escape) (32, 34),
papE fimbriae of E. coli (host cell recognition
and adhesion) (25), ABC transporters (resistance to
bacitracin [39] or pristinamycin [2]).
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FIG. 2.
Properties of L. monocytogenes
F.4565-specific fragments differ from those common to tester and
driver. Functional categories are as assigned by homology (for further
details, see Table 1).
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Hybridization patterns of L. monocytogenes
F.4565-specific fragments with genomes of different L. monocytogenes strains and species.
In order to obtain
information on the occurrence of L. monocytogenes
F.4565-specific fragments in the genomes of L. monocytogenes strains of the same or other serotypes, we hybridized the arrays with
whole genome probes of 14 L. monocytogenes strains
(corresponding to 12 independent isolates; Table
2). According to hybridization patterns,
the 4b strains were subdivided into two groups (4b-I and 4b-II), 1/2a
strains were divided into three groups (1/2a-I, 1/2a-II, and 1/2a-III),
serotype 3b and 1/2b strains grouped together, and the 1/2c strain
grouped together with the 1/2a-III strain (Table 2 and Fig.
3). Thus, according to the number of
shared hybridization signals, 4b strains appeared more related to 3b and 1/2b strains than to 1/2a and 1/2c strains. These results reflect
the established division of L. monocytogenes strains into two groups (one comprising 1/2a and 1/2c and one comprising the 4b,
1/2b, and 3b strains) on the basis of multilocus enzyme
electrophoresis, ribotyping, pulse-field gel electrophoresis, and
restriction enzyme analysis (6, 8, 21, 38) and,
additionally, into subgroups of 1/2a and 4b strains by more resolving
methods, such as PCR-restriction enzyme analysis of the
in1A-in1B region (14) or sequencing of the
in1B gene (15).
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TABLE 2.
Hybridization patterns of 39 L. monocytogenes
F.4565-specific gene fragments with DNA from different L. monocytogenes strains
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FIG. 3.
Summary of hybridization profiles of strain
F.4565-specific fragments. Black boxes indicate positive, grey boxes
indicate weak, and white boxes indicate negative hybridization signals.
The number of hybridizing fragments is indicated below each column, and
weakly hybridizing fragments are shown in parentheses. The GC content
of the fragments is indicated; in the case of not fully sequenced
fragments, the GC contents of the left and right parts of the fragment
are given separately. Asterisks indicate fragments present in the EGD
genome with high sequence diversity. Abbreviations: methyltransf.,
methyltransferase; surf. antig. expo.; surface antigen expression;
transp., transporter.
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Apart from controls, no significant hybridization signal after
low-stringency washing was seen with genomic DNA of
E. coli,
B. subtilis (data not shown), or
Listeria grayi,
the member of
the genus
Listeria most distantly related to
L. monocytogenes (Fig.
1B). However, DNA from the animal
pathogen
L. ivanovii and
from nonpathogenic
L. innocua strongly cross-hybridized to five
and nine fragments,
respectively, only two of which were common
(Fig.
1B and Fig.
3). A
second
L. innocua strain showed an identical
hybridization
behavior (data not shown). These latter results
raise the possibility
that the genomes of at least three different
species within the genus
Listeria may have a mosaic-like composition
with respect to
these
genes.
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DISCUSSION |
Five to 6% of DNA segments randomly picked from libraries from
the unsubtracted genome of L. monocytogenes strain F.4565
did not hybridize to strain EGD DNA, indicating that about 5% of the F.4565 genome is not present in strain EGD. Physical genomic maps are
available for L. monocytogenes strains EGD (serotype 1/2a) (49) and Scott A (serotype 4b) (23) and have
yielded genome sizes of 3,000 ± 50 and 3,210 ± 60 kb,
respectively. Assuming that strain F.4565 has a genome size similar to
that of strain Scott A, which has the same serotype, about 150 to 190 kb of its DNA would be absent or substantially different from the
genome of strain EGD. With an average RsaI fragment size of
0.8 kb, we would thus expect about 200 differential fragments to be
present in the F.4565 genome. Of these, we have isolated 39 fragments corresponding to about one-fifth of the theoretically expected number.
(The latter may explain why we failed to isolate the genes gtcA and gltAB, which are known to be present in
serotype 4b but not in serotype 1/2a strains [30, 40].)
Our results are in line with interstrain genome comparison data
available for other bacterial species. E. coli strains with different pathogenic potential or different host ranges were found to
vary by 300 to more than 1,000 kb (9, 12, 37), and the genomes of different Salmonella sp. serovars can differ by
greater than 900 kb (36). Recent pairwise whole-genome
comparisons of Helicobacter pylori and Chlamydia
isolates showed that strain-specific genes tend to cluster in distinct
genomic regions of about 50 to 160 kb termed plasticity zones (3,
43), while E. coli O157:H7 and E. coli
K-12 differ by hundreds of islands of DNA that are apparently
introgressed into a shared sequence backbone (37). Also,
the extent of genome diversification varies: while different
Chlamydia isolates showed little strain-specific genome material (43), the two unrelated H. pylori
isolates each had 6 to 7% strain-specific genes (3), and
the E. coli O157:H7 and K-12 isolates had about 25 and 12%
strain-specific genes, respectively (37).
H. pylori isolates seem to differ mainly in components of
restriction modification systems, insertion sequences, and DNA repeats (1, 3), while the E. coli isolates do not seem
to differ by functionally distinct groups of genes (37).
This is in contrast to the situation in L. monocytogenes,
where 41% of the strain-specific genes exhibited homology to proteins
exposed on the bacterial surface, while for 54% of the genes, no
function on the basis of sequence similarity could be predicted (Fig.
2). We found only one DNA segment with homology to a restriction
modification system component: a methyltransferase from L. lactis sharing homology with the target recognition domain of
M · Sau3A (47). The presence of this DNA
fragment in the genomes of the different L. monocytogenes strains used by us (Table 2 and Fig. 3) correlated with resistance of
their DNA to Sau3A restriction (data not shown). Resistance to Sau3A restriction has previously been reported as a
prevalent trait of epidemic-associated L. monocytogenes
serotype 4b strains (50).
Based on the sequences of the large and small intergenic spacer regions
between the 16S and 23S rRNA genes, L. monocytogenes strains
of different serotypes are more related to each other than to L. innocua (20). The nucleotide diversity of the
sigmaB and iap genes accessible in the databases
is consistent with this interpretation (data not shown). Within the
genus Listeria, the nonpathogen L. innocua is the
species most closely related to L. monocytogenes, while the
animal pathogen L. ivanovii and nonpathogen L. grayi are more distantly related (20). Surprisingly,
we found that two partially overlapping sets of nine and five L. monocytogenes F.4565-specific fragments strongly hybridized with
the genomes of L. innocua and L. ivanovii,
respectively (Fig. 1 and 3). Also, the other serotype 4b-specific genes
isolated to date, gtcA and gltAB, which are loci
involved in cell wall teichoic acid-associated surface antigen
expression, cross-hybridize to certain strains of L. innocua
(29, 30, 40). While some of these fragments may have been
lost by some lineages of L. monocytogenes, others may have
been acquired by lateral gene transfer. Thus, our data raise the
possibility that lateral gene transfer could play a role in the
diversification of L. monocytogenes strains. Indeed, this
has recently been substantiated for gtcA (29).
Repeat regions are preferential subjects of lateral gene transfer
events. Many of the F.4565-specific fragments had homologies to
repetitive protein elements (Table 1). Repeats facilitate recombination
events, which are thought to occur about 50-fold more frequently than
point mutations and therefore to have a much higher impact on bacterial
genome diversification, in both gram-negative (E. coli,
Neisseria meningitidis) and gram-positive
(Streptococcus pneumoniae) bacteria (22). In
particular, the set of fragments cross-hybridizing with L. innocua shows homology to multidomain, repeat-containing surface
proteins such as internalins and chitinases (Fig. 3). Lateral gene
transfer has been implicated in the generation and diversification of
chitinases (especially the cadherin-like domain) (35) and
proteins containing leucine-rich repeat short sequence motifs thought
to be involved in specific protein-protein interactions
(27). Moreover, mosaic internalin genes have been described (41). Consistent with this possibility, a large
number of transducing Listeria phages have been isolated
recently (24). It is also interesting that one of the
nondifferential control fragments (no. 104B) shows some homology to a
B. subtilis gene involved in DNA uptake (10).
Our hybridization analysis with arrays harboring the 39 F.4565-specific
fragments against genomes of different L. monocytogenes strains suggests that L. monocytogenes strains can be
subtyped by genomic differences (Fig. 3). Because of its higher
resolution, this approach is superior to serotyping for serotype 1/2a
and 4b strains. Thus, genomic differences could be exploited for typing L. monocytogenes strains by combinatorial PCRs or by genome
hybridizations to premanufactured typing arrays.
Taken together, we have shown that epidemic L. monocytogenes
strains differ substantially from the sequenced prototype strain of
L. monocytogenes. Many of the isolated gene fragments have homology to bacterial surface components and are therefore likely to
confer traits that provide selective advantages in the environment. While this may be a general phenomenon applying to strain
diversification of virulent as well as nonvirulent bacterial strains,
our results nevertheless provide a good starting point to search for
epidemic-trait-associated genes. For example, the complete set of genes
specifically present (or absent, since pathogenicity traits may also be
due to lack of specific genes [36]) in an
epidemic-trait-associated strain can now be identified and screened
against a large number of genomes from different L. monocytogenes isolates and Listeria species. Identification of genes consistently present or absent in
epidemic-associated L. monocytogenes strains will open the
way for mutational and functional analyses to address the question of
whether indeed there is a molecular basis for the increased pathogenic
potential of epidemic L. monocytogenes strains.
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ACKNOWLEDGMENTS |
We are grateful to Jim McLauchlin, London, for providing many of
the bacterial strains; to Ali Hemmati-Bivanlou, New York, N.Y., and
Chris Wilson, Cambridge, England, for technical advice concerning DNA
arrays; to our colleagues from the Institute for Genetics, Uli
Böhm, Thorsten Klamp, and Gloria Esposito, for advice with
subtractive techniques; to Rita Lange for DNA sequencing; and to Olga
Vites and Jonathan Howard for stimulating criticism throughout the
work. We thank the European consortium involved in sequencing the
L. monocytogenes EGD-e genome for the possibility of
analyzing the complete genome sequence prior to its release, especially
P. Cossart, Paris, France, who made this possible, and P. Glaser,
Paris, France, who kindly carried out the analysis and discussed the
results with us. Many thanks also to colleagues whose comments have
helped to improve the manuscript.
This work was supported by the Deutsche Forschungsgemeinschaft through
Sonderforschungsbereich 274.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute for
Genetics, University of Cologne, Zuelpicher Str. 47, D-50674 Cologne, Germany. Phone: 49 221 470 4857. Fax: 49 221 470 5172. E-mail: Christine.Kocks{at}uni-koeln.de.
Editor:
B. B. Finlay
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Infection and Immunity, June 2001, p. 3972-3979, Vol. 69, No. 6
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.6.3972-3979.2001
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Abstract
Introduction
