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Journal of Bacteriology, June 2001, p. 3811-3815, Vol. 183, No. 12
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.12.3811-3815.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Insertion Mutagenesis of wca Reduces Acid and Heat
Tolerance of Enterohemorrhagic Escherichia coli
O157:H7
Ying
Mao,
Michael P.
Doyle, and
Jinru
Chen*
Center for Food Safety and Department of Food
Science and Technology, University of Georgia, Griffin, Georgia
30223-1797
Received 27 December 2000/Accepted 30 March 2001
 |
ABSTRACT |
Strains of enterohemorrhagic Escherichia coli (EHEC)
serotype O157:H7 produce under stress copious amounts of
exopolysaccharide (EPS) composed of colanic acid (CA). Studies were
performed to evaluate the association of production of CA with survival
of EHEC under adverse environmental conditions. A CA-deficient mutant, M4020, was obtained from a CA-proficient parental strain, E. coli O157:H7 W6-13, by inserting a kanamycin resistance gene
cassette (kan) into wcaD and wcaE,
2 of the 21 genes required for CA biosynthesis. M4020 was defective in
CA production as determined from the ratio of uronic acid to protein
(UA/P) of cells grown from 1 to 4 days at 25°C on minimal glucose
agar (MGA), MacConkey agar, and sorbitol-MacConkey agar, and by colony
morphology on MGA. The results of stress treatment revealed that M4020
was substantially less tolerant to acid (pH 4.5 and 5.5) and heat (55 and 60°C) in comparison to W6-13, indicating that CA confers on
E. coli O157:H7 a protective effect from the environmental
stresses of acid and heat.
| |
TEXT |
Escherichia coli O157:H7
is a serious foodborne pathogen, causing life-threatening maladies
including hemorrhagic colitis, hemolytic-uremic syndrome, and
thrombotic-thrombocytopenic purpura (22, 25, 30). Although
outbreaks of E. coli O157:H7 infection are frequently
associated with eating undercooked ground beef, a variety of other
foods, including dry and acidic foods, also have been implicated as
vehicles of infection (4, 5). Outbreaks associated with
highly acidic foods are of particular concern because acidic conditions
are normally considered sufficient not only to inhibit the growth of
but also to kill most foodborne pathogens. Hence, the tolerance of
E. coli O157:H7, which has a low infectious dose, to acidic
foods compounds the serious nature of this bacterium as a foodborne pathogen.
Many strains of E. coli O157:H7 are substantially more
tolerant to adverse environmental conditions than many nonpathogenic E. coli strains (2, 17), with the pathogen
surviving for prolonged periods of time in a range of foods under
various adverse conditions (20, 26). E. coli
O157:H7 survived acid stress in 1.5% (vol/vol) acetic acid (pH 4.0) at
37°C for 15 min, whereas a nonpathogenic strain of E. coli
did not survive under the same conditions (32). In a
laboratory study, E. coli O157:H7 survived the drying
process used to make venison jerky under several time and temperature
combinations, including up to 10 h at 62.8°C (15). Viable E. coli O157:H7 was detected after 158 days of
ripening in cheddar cheese made with milk inoculated with
103 CFU/ml (24). While the endurance of
E. coli O157:H7 to environmental stress has been well
documented, the molecular mechanisms affecting such tolerances are
poorly understood.
Many factors contribute to bacterial resistance to environmental
stress. Among these are cell surface structures and appendages which
provide the firstline of defense for a bacterium. As with many other
members of the Enterobacteriaceae, E. coli secretes a
variety of exopolysaccharides (EPS), including colanic acid (CA). CA
contains L-fucose, D-glucuronic acid,
D-glucose, D-galactose, and pyruvate and forms
a thick mucoid matrix on cell surfaces (8, 10, 23, 31). CA
biosynthesis in E. coli K-12 is encoded by the
wca gene cluster, which includes 21 open reading frames (ORFs) (28). Among these ORFs, wcaC and
wcaE encode putative CA glycosyl transferases, and
wcaD produces a putative CA polymerase. These three genes
are located on the upper portion of the giant CA operon and are
involved in sequential transfer of the component sugars of CA and
assembly of the CA polymer.
Junkins and Doyle examined 27 E. coli O157:H7 strains and
determined that 67% (18 of 27) formed mucoid colonies on
sorbitol-MacConkey (SMAC) agar during prolonged incubation at ambient
temperature (14). Four of the five nonslime-forming
strains on SMAC agar did become mucoid on media containing a higher
concentration of sodium chloride. Further analysis revealed that CA was
the principal component of EPS produced by E. coli O157:H7.
However, no association has been made with CA and its influence on
survival of E. coli O157:H7 under conditions of stress.
In the study presented here, a CA-deficient mutant was constructed by
inserting a kanamycin resistance gene cassette (kan) into
the wca operon of the E. coli O157:H7 genome. CA
production by the parental and mutant strains was determined by
measurement of the uronic acid/protein (UA/P) ratio (14).
Heat or acid treatments were subsequently applied to the parental and
mutant strains to determine the survival characteristics of the
isogenic pair.
Construction of CA-deficient mutant.
More than a hundred
E. coli O157:H7 strains from our laboratory collection were
screened on SMAC agar for EPS-forming colonies, a
presumptive indication of CA production. CA-producing E. coli O157:H7 W6-13 was eventually selected as a parental strain
because of its production of copious amounts of EPS. Two PCR fragments (upstream and downstream) were amplified from the W6-13 chromosome using the oligonucleotide primer pairs, P1 and P2
(5'-AAAGCTTAAACCGGACGTCACT-3' and
5'-GAATTCTCCTCCACACCATGCCAAT-3';
EcoRI site underlined) and P3 and P4
(5'-AAAGAATTCCAAAGGCATTGC-3' and
5'-TTCGCGTCAGCACACAATTC-3'; EcoRI site
underlined), respectively. The PCR primers were derived based on
GenBank sequences within wcaC, wcaD, and wcaE of
E. coli K-12 (GenBank accession no. U38473). Both primers 2 and 3 had an artificial EcoRI site near the 5' end.
Following PCR, the two amplified DNA fragments were digested with
EcoRI and then ligated to each other to create an
EcoRI restriction site. This fragment with the
EcoRI site was subsequently cloned into plasmid vector pGEM-T (Promega Co., Madison, Wis.) to generate recombinant plasmid pYM37 (wca
DE). The
EcoRI restriction site on pYM37 was used to receive an
EcoRI-EcoRI kanamycin resistance gene cassette
which was PCR amplified from pNEO (Amersharm Pharmacia Biotech, Inc., Piscataway, N.J.) and restricted with EcoRI. The resulting
plasmid was designated pYM3720 from which the
wcaDE::kan fragment was amplified with
primer pair P1 and P4. Amplified
wcaDE::kan fragment was introduced into
E. coli O157:H7 W6-13 by electroporation. Recombinant
colonies were selected on Luria-Bertani agar supplemented with
kanamycin (100 µg/ml). One of the colonies, M4020 (W6-13 wcaDE::kan), was isolated. The site of
insertion was confirmed by PCR amplification and Southern hybridization
with a kanamycin resistance gene probe. Insertion mutagenesis abolished
the function of wca, whereby M4020 became defective in CA production.
wcaC, wcaD, and wcaE of E. coli
O157:H7 were sequenced using an ABI Prism PCR Sequencer (Molecular
Genetics Instrumentation Facility, University of Georgia). The
sequences were deposited in GenBank (accession no. AF320069). Sequence
analysis revealed that these genes share 98% homology with the same
genes of E. coli K-12.
Quantification of CA production.
CA produced by E. coli W6-13 and M4020 was quantified according to a procedure
previously described by Junkins and Doyle (14). The UA/P
ratio, defined as micrograms of uronic acid per milligram of protein,
was used to estimate CA production of both strains on minimal glucose
agar (MGA), MacConkey (MAC) agar, and SMAC agar. While colonies of
W6-13 and M4020 were visible on agar plates after 16 h of
incubation, development of a mucoid appearance by W6-13 required at
least 24 h of incubation at 25°C. UA/P ratios of the same strain
varied substantially among media used for cultivating the cells.
However, CA production increased with incubation time, with the
greatest amount being produced at day 2 on MGA and MAC agar and at day
4 on SMAC among the various days of incubation studied (Fig.
1A). Colonies became extremely mucoid due
to overproduction of CA (Fig. 1B). Greater amounts of CA were observed
when W6-13 was grown on MGA than on complex media, such as MAC agar,
SMAC agar (Fig. 1A), and MGA containing 0.5% Casamino Acids (wt/vol) (data not shown). This agrees with previous reports that CA production in E. coli K-12 is enhanced when cells are grown under
less-than-optimal conditions for growth, such as in presence of low
temperature, high salt concentrations, or high concentrations of
utilizable carbon sources (18, 21, 27). Although many
strains of E. coli O157:H7 produce CA, the amounts produced
can vary greatly. Furthermore, CA production occurs when cultures grow
at 25°C rather than at 37°C, which may be a feature to provide
bacteria with a means to respond to stress and better survive
conditions outside their human and animal hosts.
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FIG. 1.
UA/P ratio of E. coli O157:H7 W6-13 and its
CA-deficient mutant M4020 when grown on different media, and CA
production by colonies of W6-13 and M4020. (A) UA/P ratio of W6-13 and
M4020 grown at 25°C for 1, 2, and 4 days on MGA, MAC agar, and SMAC
agar. (B) CA production of E. coli O157:H7 W6-13 (left) and
M4020 (right) when grown on MGA at 25°C for 4 days.
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|
CA production by
E. coli O157:H7 W6-13 was also compared to
that of the nonpathogenic
E. coli K-12 strain ZK2686 which
is
known to produce CA (
6). The K-12 strain produced
colonies
with much less EPS than by
E. coli O157:H7 W6-13
under the same
conditions on MGA, supporting our earlier hypothesis
that the
overproduction of CA may play a more important role in greater
resistance to environmental stresses by certain pathogenic than
nonpathogenic
microorganisms.
Protective effect of CA on acid and thermal tolerance.
In this
study, W6-13 and M4020 were subjected to acid treatment with pH levels
ranging from 4.5 to 6.5 and to heat treatment at 55 and 60°C to
evaluate the effect of CA expression on acid and thermal tolerance. For
the acid treatment, W6-13 and M4020 were inoculated onto MGA and
incubated at 25°C for 24 h. Colonies were removed with minimal
glucose broth (MGB), and cell suspensions of W6-13 and M4020 were
inoculated (1%) (vol/vol) into fresh MGB containing 5% Casamino Acids
adjusted to pH 4.5, 5.5, and 6.5 with 0.1 M HCl. The cultures were
incubated at 37°C, and viable counts of E. coli O157:H7
were determined at selected intervals. Approximately 108
CFU/ml of cell suspension of each strain was heated at 55 and 60°C
for the thermal inactivation studies. Viable counts of E. coli O157:H7 were determined by sampling at selected time
intervals, plating bacteria on MGA containing 0.5% Casamino Acids and
incubating the plates at 37°C for 24 h. The D-value, which is
defined as the time at a specific temperature that is required to
inactivate 1 log of the bacterial population (12), was
calculated by linear regression analysis.
Although insertional mutagenesis abolished the function of
wca, mutant M4020 at 37°C grew at a similar rate and
survived as
well as its isogenic parental strain W6-13. During acid
treatment
with pH levels ranging from 4.5 to 6.5, both W6-13 and M4020
grew
in MGB at a similar rate at pH 6.5 (Fig.
2A); however, the growth
of M4020 lagged
slightly at pH 5.5 (Fig.
2B). The growth of W6-13
at pH 4.5 was not
affected substantially compared to growth at
pH 5.5 or 6.5; however,
the growth of M4020 was completely inhibited
at pH 4.5 (Fig.
2C).
Similarly, M4020 was more susceptible than
W6-13 to sublethal
temperatures as reflected by significantly
lower D-values at 55 and
60°C (Table
1).
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FIG. 2.
Viability of E. coli O157:H7 W6-13 ( ) and
M4020 ( ) at 37°C in MGB containing 0.5% Casamino Acids and
adjusted to pH 4.5, 5.5, or 6.5 with 0.1 M HCl.
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TABLE 1.
Effect of CA on thermal tolerance of E. coli
O157:H7 wild-type strain W6-13 and its CA-deficient mutant M4020 in
MGB containing 0.5% Casamino Acids
|
|
It has been suggested based on the results of previous studies that CA
expressed on cell surfaces may simply act as a physical
barrier to
protect cells from hostile environmental conditions,
such as enabling
cells to retain water in a dry environment (
1,
18,
21,
27). It was determined in another study that
E. coli
O157:H7 cells are rapidly inactivated by a rapid accumulation
of
protons (
13). In an acidic environment, the lower the pH,
the greater the concentration of protons. CA confers a strong
negative
charge to the cell surface which, we suggest may serve
as a buffer by
neutralizing protons at the cell surface, whereby
preventing positively
charged chemical groups from accumulating
on cell envelopes and from
penetrating into cells. We further
hypothesize that the amount of CA on
cell surfaces determines
the buffering capacity of cells. When cells
lose their ability
to produce CA, cell surfaces become less negatively
charged and
thereby have reduced buffering capacity. When negatively
charged
cell surfaces are neutralized, protons will accumulate and
enter
cells freely. Such a change in intracellular pH will impair cell
metabolism, causing cell
death.
Studies revealed that the expression of CA is regulated by several
direct and indirect mechanisms (
9,
16,
19). The
transcription of the
wca operon is positively regulated by
RcsA,
RcsB, and RcsF and negatively regulated by ATP-dependent protease
Lon (
3,
11,
16,
29). RcsC can be activated by
environmental
stimuli and both positively and negatively regulates CA
biosynthesis
(
29,
31). RcsC and RcsB act as a sensor and
an effector, respectively,
in a two-component regulatory system
(
9,
29,
31). Several
heat shock proteins, including Dnak
(Hsp70), GrpE, and DnaJ, also
influence CA biosynthesis
(
16). A recent study revealed that
DjlA, a member of the
DnaJ family, is a DnaK cochaperone, and
the induction of the colanic
acid requires this DnaK-DjlA interaction
to stimulate the RcsB-RcsC
two-component signaling system in
E. coli (
7).
Although CA on cell surfaces may act as a physical
barrier and affect
heat conductivity, thereby preventing heat
from reaching cells readily,
the influence of several heat shock
proteins on the regulation of CA
induction could further suggest
a role for CA in the heat response of
E. coli O157:H7.
Results of this study demonstrated that colanic acid is inducible in
E. coli O157:H7, mostly by certain environmental stimuli,
and that the induction of colanic acid has a substantial protective
effect on the pathogen's acid and thermal tolerance. However,
there
are likely other factors of
E. coli O157:H7 that confer
to
the pathogen additional protection against stressful environmental
conditions. The mechanism of stress response in
E. coli
O157:H7
is complex, and other factors in addition to the induction of
colanic acid are likely involved in conferring tolerance to the
pathogen under stress-imposed conditions. Although there is limited
information addressing the association of CA production with
pathogenicity
(
1), a recent report suggests a role for
colanic acid in the
architecture of biofilms in the K-12 strain
(
6). Additional
studies are under way to further elucidate
the role of CA in the
pathogen's exceptional tolerance to other forms
of environmental
stress, as well as in bacterial attachment and biofilm
formation.
| |
ACKNOWLEDGMENTS |
We thank Paul Danese in R. Kolter's lab, Harvard Medical School,
for providing strain E. coli K-12 ZK2686.
This research was supported in part by a grant from the U.S. Department
of Agriculture for the Alliance for Food Protection.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for Food
Safety, University of Georgia, 1109 Experiment St., Griffin, GA
30223-1797. Phone: (770) 412-4738. Fax: (770) 229-3216. E-mail:
jchen{at}cfsqe.griffin.peachnet.edu.
| |
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Journal of Bacteriology, June 2001, p. 3811-3815, Vol. 183, No. 12
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.12.3811-3815.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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