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Yersinia enterocolitica
is a human pathogen and a causative agent of gastroenteritis which is
transmitted by the oral-fecal route (8). As is frequently
true of gastrointestinal parasites, Y. enterocolitica is
capable of prolonged survival outside a human host, in soil and water
at temperatures as low as 4°C (9). Consequently, it has
adapted to grow in two environments, in nutrient-limited natural
environs at ambient temperature and in a generally nutrient-rich host
at 37°C. The adaptation to a dual lifestyle is reflected by
temperature regulation of Y. enterocolitica metabolism
(prototrophic at <30°C, auxotrophic at 37°C), motility, O-antigen
structure, and virulence genes (9, 13, 34, 37).
Expression of the virulence genes ail, inv,
yst, yadA, and myfA; the
ysc type III secretion system; and the yop genes
is affected by temperature (11, 13, 22, 25, 29, 34, 37-39).
Notably, the yst and inv virulence genes are
expressed poorly at 37°C unless the bacteria are grown under
conditions of greater osmolarity or lower pH, respectively (34,
37). Motility is also affected by temperature: Y. enterocolitica is motile at low temperatures (<30°C)
(10). Interestingly, characterization of flagellar
regulation has demonstrated that some Y. enterocolitica
flagellar genes are regulated in response to temperature, yet others
are not. Increasing the temperature above 30°C quickly repressed
transcription of the fleABC class III flagellar genes in
vitro (23). The flagellar sigma factor (
F or
FliA) directs transcription of class III genes, and fliA
itself is temperature regulated (24). However, transcription
of the class II flagellar genes flhBAE was unaffected by a
shift to 37°C (14). By analogy to Escherichia
coli and salmonellae, class II flagellar proteins form a
transmembrane structure which serves as the flagellar type III
secretion apparatus (1). Considering that a functional
flagellum is not made at 37°C, it is unclear why some flagellar genes
would be expressed unless (i) this partial flagellar structure served
another purpose or (ii) being able to quickly complete the partial
flagellum is advantageous in case conditions change. A recent study
suggested that the flagellar basal body (encoded by class II genes) is
able to function as a type III-like secretion apparatus, and this may
account for the function of this structure at 37°C in the absence of
a flagellum (49).
Previous experiments have established that the Y. enterocolitica phospholipase (YplA) is a virulence factor that
influences the ability of Y. enterocolitica strain 8081v
(39) and its derivatives to colonize tissues and to induce a
more vigorous inflammatory response (41). To better
understand the role of this phospholipase in pathogenesis, we
characterized the pattern of yplA expression in Y. enterocolitica strains derived from 8081v (Table
1) in response to environmental
conditions, especially those conditions which affect the expression of
other known virulence factors. In addition, we examined the possibility
that yplA is regulated as part of the flagellar regulon
because it has a potential flagellar F promoter and it
has been demonstrated that the phospholipase is secreted through the
flagellar type III secretion system (49).
Sequence analysis of the yplAB promoter region.
The nucleotide sequence was scanned for potential regulatory sequences
that might influence yplA expression; this required additional sequence information upstream of yplAB (GenBank
accession no. AF0678496). The template pDHS20 (41) was
purified by the alkaline lysis method (31), sequenced using
the ABI Prism Bigdye Terminator Cycle Sequencing System (Perkin-Elmer),
and read on an ABI Prism 377 DNA Sequencer (Nucleic Acid Chemistry
Laboratory, Washington University). Sequence analysis using the
Wisconsin Sequence Analysis Package (GCG, Madison, Wis.) indicated that the arrangement of genes and their predicted amino acid sequences are
very similar to those in the phlAB locus of Serratia
liquefaciens (41). S. liquefaciens phlAB
encodes a phospholipase A1 and its putative accessory
protein, respectively (18, 19). Located 221 bp upstream of
yplAB is another open reading frame, yplX, transcribed from the same strand as yplAB. The predicted
amino acid sequence of Y. enterocolitica yplX is 90%
identical (over 111 residues) to that of similarly placed
orfX found in S. liquefaciens (17), as
well as 85 and 83% identical to hypothetical proteins encoded in
E. coli and Haemophilus influenzae (6,
15). All of these small proteins share significant identity (80%
identity over 61 residues) with the acetyltransferase domain of
E. coli pyruvate formate lyase (formate acetyltransferase)
(48). Further examination of the Y. enterocolitica nucleotide sequence indicated that divergently
transcribed genes flank the hypothetical acetyltransferase yplX and yplAB, suggesting that the maximal
possible transcriptional unit is restricted to yplXAB.
Examination of the upstream regions for potential regulatory sequences
such as promoters and regulatory protein binding sites has identified
several regions of interest. Preceding yplX by 113 bp is a
sequence with similarity to the binding site consensus for Fnr
(46). Fnr is a global activator of genes expressed under anaerobic conditions that is itself oxygen sensitive (4).
Examination of the intergenic sequence between yplX and
yplA had confirmed a potential binding site for flagellar
sigma factor F (FliA) (21, 41) and Crp
(7). The potential F and Crp binding sites
are located 55 and 92 bp upstream of yplA, respectively.
FliA (F) is the alternate sigma factor that directs
transcription of class III flagellar genes. Crp is a global regulator
that responds to cytoplasmic cyclic AMP (cAMP) levels (7).
Comparison of the intergenic sequences of Y. enterocolitica
and S. liquefaciens determined that regions with the highest
identity encompass the potential regulatory sites. Matches to Fnr,
cAMP-Crp, and F binding sites were found similarly
positioned in the S. liquefaciens nucleotide sequence
(17). Furthermore, expression of Serratia phlAB
was shown to be dependent on functional cya (adenylate
cyclase) and flhDC genes in E. coli
(17). The pattern of phlAB regulation in S. liquefaciens was consistent with catabolite repression (inhibition by glucose) and was shown to require functional flhD
(16, 17).
Environmental signals affecting yplAB expression.
The Y. enterocolitica reporter strain YEDS16 was generated
with yplA::lacZY located in the
appropriate chromosomal environment in single copy to reflect
expression of yplA (Fig. 1).
YEDS16 is a merodiploid with a fully intact yplX yplAB
(yplXAB) region with another copy of yplXA'
transcriptionally fused to lacZY. To generate a
transcriptional fusion in the Yersinia chromosome, pDHS45
(yplA'::lacZY) was mated into Y. enterocolitica strain JB580v (26) as previously
described (42). pDHS45 replicates from an R6K origin; thus,
it can only be stably maintained if the plasmid integrates into the
chromosome by homologous recombination (27, 42). Y. enterocolitica transconjugants were selected on Luria broth (LB)
supplemented with nalidixic acid at 20 µg/ml and chloramphenicol at
25 µg/ml. This strain, YEDS16, was found to be 
-galactosidase and
phospholipase positive on plates (1% tryptone agar or MacConkey agar
base) incubated at 26°C supplemented with
5-bromo-4-chloro-3-indolyl--D-galactoside at 20 µg/ml
or 0.2% egg yolk lecithin and 1 mM CaCl2, respectively
(data not shown); under the same conditions, 8081v is also positive on
lecithin plates (41). This is consistent with the functional
phospholipase promoter being duplicated and driving lacZY
and yplAB expression in YEDS16.
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FIG. 1.
Integration of pDHS45 into the Y. enterocolitica chromosome (above) with resolution of the
homologous recombination event (below) to generate a chromosomal
reporter fusion. The coding regions of the phospholipase,
yplA, its putative accessory protein, yplB, and
the hypothetical acetyltransferase, yplX, are depicted as
boxes. In the boxes, arrows indicate the direction of transcription and
the positions of predicted regulatory protein binding sites are
indicated by labeled arrowheads in the normal locus diagrammed at the
top. The suicide plasmid pDHS45 carries a 2.2-kb
XbaI/NheI fragment from pDHS21 that encompasses
sequences 5' to yplA and was ligated into the unique
XbaI site adjacent to the intact lacZY genes
(with a ribosomal binding site) on the suicide vector pFUSE
(3). The functional yplAB locus is encoded within
pDHS21 (this study; yplAB is in the opposite orientation
within previously described plasmid pDHS20 [41]). The
yplAB region diagrammed is present in strains YEDS16,
YEDS18, and YEDS29. Plasmid sequences are indicated by the broken line;
the solid line indicates Yersinia sequences. The correct
insertion of yplA'::lacZY (on pDHS45)
was confirmed by Southern analysis (45) of SphI
and EcoRI digests of chromosomal DNA purified as previously
described (32) using a yplA fragment as a probe
(data not shown). A 662-bp fragment of yplA was amplified by
PCR using the primers 5' AAGAACTCATCCGATGTG 3' and 5'
AGTGCATCGACCAAATGCC 3'.
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An initial observation indicated that levels of phospholipase activity
produced by Y. enterocolitica decreased when salt was added
to the medium (when assayed on lecithin plates). Consistent with this
observation, comparison of the -galactosidase activity produced by
overnight cultures of the reporter strain, YEDS16, grown in either 1%
tryptone broth, 1% tryptone with 200 mM NaCl, or LB (which contains
171 mM NaCl) determined that yplA expression was greatest in
the 1% tryptone broth cultures (data not shown). -Galactosidase
assays were performed and values were calculated as previously
described (35). This finding suggests that the inhibitory
effect of salt on YplA activity is exerted at the transcriptional level. Thereafter, all experiments used Y. enterocolitica
cultures grown in 5 ml of 1% tryptone broth based media under standard conditions, i.e., incubation in a culture tube on a wheel at 26°C for
8 h, unless otherwise indicated. Cultures for determination of
yplA expression were inoculated from overnight LB cultures, conditions known to produce very low levels of yplA
expression. Thus, residual -galactosidase activity from the
overnight cultures would not be detected in the samples assayed. The
inhibitory effect of NaCl was further examined to distinguish among
several possible environmental cues which might be causing the effect:
NaCl concentration, ionic strength, and osmolarity (Fig.
2A). The effects of ionic strength versus
osmolarity were tested in 1% tryptone broth supplemented with
increasing salt (0 to 300 mM KCl or NaCl) or rhamnose (0 to 600 mM)
concentrations, as indicated. The level of -galactosidase activity
(yplA expression) decreased similarly with increasing concentrations of NaCl and KCl, but only the highest concentration of
rhamnose (600 mM) caused significant repression. Thus, the environmental cue triggered by NaCl seems to be either chloride concentration or ionic strength generally rather than osmolarity, although high osmotic strength is also apparently inhibitory.
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FIG. 2.
Effects of ionic strength and osmolarity (A), growth
phase and temperature (B), and pH (C) on -galactosidase activity
production by yplA'::lacZY present in
Y. enterocolitica YEDS16. (A) Cultures were grown at 26°C
for 8 h in 1% tryptone supplemented with the concentrations of
NaCl ( , solid line) and KCl (×, dotted line) indicated on the
bottom x axis and the concentrations of rhamnose ( ,
hashed line) indicated on the top x axis. -Galactosidase
activity is shown on the y axis in Miller units. The
OD600 of the cultures in these media were comparable after
8 h, except for those with 400 or 600 mM added rhamnose, in which
YEDS16 grew slower. (B) Cultures were grown in 1% tryptone at 26°C
( , solid line) and at 37°C (, hashed line) for 29 h, and
samples were taken initially every 3 h. The top graph plots the
-galactosidase activity on y axis (Miller units) versus
time (h) on the x axis. The bottom graph depicts bacterial
growth over time for the same cultures. OD600 is plotted on
the y axis, and time is on the x axis. (C) A
series of buffered 1% tryptone broth cultures (50 mM buffer, pH range
of 5.0 to 9.5) were inoculated from the same overnight YEDS16 LB
cultures and incubated for 8 h at 26°C. The final pH of the
buffered media was determined and found not to change during the course
of the experiment. The OD600 of the cultures in the
buffered media were comparable after 8 h, except for that of the
pH 9.5 medium, which had a lower OD600. Consequently, the
cultures in pH 9.5 medium were allowed to grow for an additional 4 h; the -galactosidase activities at 8 and 12 h were similar,
even though the 12-h culture had a greater OD600. The
standard deviation of the mean is represented as error bars for each
plotted point.
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The level of expression was monitored at various times during the
growth of YEDS16 to determine at which phase of growth yplA is most highly expressed (Fig. 2B). -Galactosidase activity peaked during mid-to-late logarithmic phase, but significant activity was
detected late into stationary phase. Induction of expression at the
transition from logarithmic growth to stationary phase has been
demonstrated for other virulence genes, including inv, yst, ail, and myfA (22, 34, 37,
38). Although the initial increase of yplA
transcription is initiated in late log phase, significant
transcriptional activity continues well into stationary phase.
Experiments examining phospholipase activity produced by Y. enterocolitica on lecithin plates established that phospholipase activity was produced at 26°C but was not detected at 37°C. There are several possible explanations for this temperature effect: yplA may not be efficiently transcribed, YplA may not be
efficiently secreted, or YplA is not active at 37°C. Active
phospholipase had been produced from a high-copy plasmid in E. coli grown at 37°C, suggesting that YplA is active at 37°C,
which is consistent with the role of YplA as a virulence factor (data
not shown). To determine whether the temperature effect was due to
transcriptional regulation, YEDS16 was grown at 37°C and assayed for
-galactosidase activity at various times during growth (Fig. 2B). In
vitro, there was no significant induction of expression in 1% tryptone
at 37°C at any phase of growth. Therefore, the lack of phospholipase
activity displayed by Y. enterocolitica in vitro at 37°C
is due to temperature regulation at the level of transcription.
Superficially, this pattern of temperature regulation is contradictory
to the role of YplA as a virulence factor. However, inv
(invasin) and yst (heat-stable enterotoxin) are also
virulence genes expressed at low temperature rather than at 37°C in
vitro (13). Yet, at slightly acidic pH or high osmolarity,
respectively, these virulence genes are expressed at 37°C in vitro
(34, 37). Moreover, invasin has been detected in yersiniae
recovered from mouse tissues 45 h after peroral inoculation in
amounts similar to those produced by the same number of bacteria
cultured at 26°C in vitro (37). Therefore,
high-temperature repression can be superseded by other conditions that
stimulate expression in the host. Alternatively, low-temperature
induction could reflect the need for the virulence factors early in the
infectious process, as has been suggested for invasin (37)
and the Y. enterocolitica urease (12). Invasin is
thought to promote initial entry through the intestinal epithelium, and
urease is thought to promote survival in the acidic environment of the
host stomach. However, a secreted virulence factor would likely be
diluted or degraded and exert little effect unless produced and
secreted within the host. Thus, it seems probable that some cue induces
expression of yplA in vivo. Future experiments will attempt
to demonstrate the presence of YplA or expression of yplA in
yersiniae infecting mouse tissues, but that is beyond the scope of this study.
A number of important Yersinia virulence genes, the
yop genes and yadA, are expressed under
low-calcium conditions at 37°C in vitro (39). Experiments
with YEDS16 demonstrated that removal of calcium by addition of 20 mM
NaC2O4 and 20 mM MgCl2 at 26°C slightly repressed expression of yplA, but addition of extra
Ca2+ (2.5 mM CaCl2) had no significant effect
(data not shown). There was no apparent effect of calcium limitation at
37°C, although Y. enterocolitica does not grow well at
elevated temperature without calcium. Virulence factors from many
pathogenic bacterial species are regulated in response to
Fe2+. Experiments in which either additional 150 µM
FeCl3 was added or available Fe2+ was removed
(chelated using 100 µM 2,2'-dipyridyl) demonstrated there was no
significant effect on transcription of yplA in response to
Fe2+ concentration (data not shown).
Y. enterocolitica survives over a large pH range (pHs 4.0 to
10.0), which is certainly beneficial as it traverses the acidic stomach
and is carried into the alkaline small intestine (5). Therefore, -galactosidase activity was assayed in cultures grown in
a series of buffered broth media (1% tryptone) inoculated in parallel.
The effect of pH was examined using 1% tryptone broth containing a 50 mM concentration of the following buffers equilibrated at the indicated
pH values: citric acid for pHs 5.0 and 6.0; HEPES for pHs 7.0 and 7.5, and TAPS
[N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid]
for pHs 8.5 and 9.5. After 8 h of incubation, bacteria were harvested for -galactosidase assays and the pH of the culture media
was tested. In no case had the pH significantly changed (>0.2 pH U)
from the original pH of the buffered media. In the pH range tested, pHs
5.0 to 9.5, the cultures reached similar cell densities (optical
density at 600 nm [OD600], 1.0 to 1.2), except for the
most alkaline medium (pH 9.5; OD600, 0.5 to 0.7). These
results suggest that yplA was efficiently transcribed at pHs
6 to 7.5 and expression peaked at pH 7.0 (Fig. 2C).
Given the identification of potential regulatory binding sites 5' to
the yplAB locus, environmental cues which affect the regulatory proteins were tested. First, the potential Fnr binding site
upstream of yplX suggests that transcription of
yplX, and perhaps yplA, might be regulated in
response to oxygen tension. Few investigators have examined the
response of Y. enterocolitica virulence gene expression at
different oxygen levels: expression of both inv and
ail is reduced under anaerobic conditions in vitro (inv is only significantly affected in rich medium at 26°C
and not at 37°C or in minimal media) (36, 37). Therefore,
the effect of aeration was determined using 5-ml cultures grown under the following conditions of increasing aeration, from least to greatest: in a culture tube on a wheel, in a slanted culture tube shaking at 250 rpm, and in a 125-ml flask shaking at 250 rpm. Static
cultures were not included for comparison because these cultures grew
slowly to a low cell density in 1% tryptone, which would not allow
meaningful comparison of promoter activities. Vigorously shaken
cultures produced a fifth of the -galactosidase activity of cultures
grown in tubes on a wheel (Fig. 3A). As
maximum expression occurs when cell density is high and free oxygen is scavenged quickly, the oxygen tension in these culture is probably low.
These results are consistent with Fnr-dependent regulation of
yplA expression. Similar conditions of low oxygen tension
occur in the gut lumen, so this condition may have relevance in the host (43).
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FIG. 3.
Effects of oxygen levels (A), added sugars (B), and
flagellar regulators (C) on -galactosidase activity production by
Y. enterocolitica YEDS16 and Western analysis of Y. enterocolitica phospholipase production from YEDS16 and flagellar
mutant Y. enterocolitica strains using anti-YplA serum (D).
(A) Five-milliliter cultures of YEDS16 were grown in 1% tryptone broth
at 26°C as follows: 1, in a 125-ml flask with vigorous aeration; 2, in a culture tube with vigorous aeration; 3, in a culture tube
incubated on a wheel. (B) Cultures were grown under the standard
conditions in 1% tryptone broth (black column) and with 20 mM added
sugars (gray columns). One percent tryptone with 20 mM Mg oxalate (MOX)
was included for comparison (striped column). (C) Effects of a
flagellar regulator mutant background on production of
-galactosidase activity from
yplA'::lacZY. Shown are results for the
'fliA mutant background, YEDS18 , with the
pTM100 vector (v) and complemented with fliA on pJB222
(fliA) and the  flhDC mutant background, YEDS29
( ), with the vector pTM100 (v) and complemented with flhDC
on pGY10 (flhDC) (dotted columns). Results for wild-type
(wt) Y. enterocolitica YEDS16 and its derivatives carrying
plasmids pTM100 (v), pJB222 (fliA), and pGY10
(flhDC) are shown as striped columns for comparison. (D)
Proteins secreted into the medium were concentrated essentially as
previously described (49), resuspended in reducing sample
buffer, and separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis essentially by the method of Laemmli (28).
The polyacrylamide gels were transferred to nitrocellulose using a
semidry transfer apparatus (Trans-Blot SD; Bio-Rad). The primary
anti-MalE-YplA serum was used at 1/2,000, followed by alkaline
phosphatase-conjugated anti-rabbit immunoglobulin G diluted to 1/30,000
(Sigma), and the Western immunoblot was developed by a
chemiluminescence assay method (ECL; Amersham).
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The identification of a cAMP-Crp binding site suggests that
yplA is regulated by catabolite repression. For members of
the family Enterobacteriaceae, the cAMP concentration varies
inversely with the growth rate and the presence of glucose both reduces cAMP synthesis and stimulates its efflux from the cytoplasm (7, 40). Therefore, 20 mM glucose, sucrose, maltose, lactose,
arabinose, galactose, or glycerol was added to 1% tryptone broth, the
cultures were incubated under the standard conditions, and then levels of -galactosidase activity were determined. The addition of 20 mM
glucose or sucrose, a disaccharide of glucose and fructose, reduced the
levels of -galactosidase activity to a quarter and approximately a
third of that observed in the 1% tryptone broth, respectively (Fig.
3B). Addition of other utilizable carbon sources at 20 mM, including
maltose, galactose, arabinose, glycerol, and lactose, did not have a
significant effect on the levels of yplA expression (Fig. 3B
and data not shown). While lactose is a disaccharide composed of
glucose and galactose, it had no significant effect on levels of
-galactosidase activity. This result is consistent with the fact
that Y. enterocolitica is unable to utilize lactose as a
carbon source. Furthermore, all of these results are consistent with
the regulation of yplA by catabolite repression.
To confirm that the patterns of transcriptional regulation determined
using the reporter fusion reflected the production of YplA protein,
Y. enterocolitica grown under various conditions was
examined by Western analysis using antiserum generated against YplA.
Polyclonal antiserum was raised in a rabbit using MalE-YplA produced
from pDHS28 in E. coli and purified as previously described (49). Confounding specificity for common bacterial antigens was removed by adsorption to acetone powders prepared from Y. enterocolitica YEDS10 (phospholipase null mutant) and E. coli containing pMal-p2 (20). In all cases, the amount
of YplA detected correlated with the levels of expression determined
using the yplA::lacZY fusion (data not shown).
yplAB expression requires the flagellar regulators
(FlhDC and FliA).
The identification of a potential flagellar
sigma factor-dependent promoter, export of YplA by the flagellar type
III apparatus, and regulation of yplA and flagellar genes in
response to temperature and ionic strength suggest that yplA
is part of the flagellar regulon. This hypothesis was tested by
introducing yplA::lacZY into Y. enterocolitica strains GY460 and JB400v, which have mutations in
the flagellar regulatory genes flhDC (50) and
fliA (2), respectively. The correct insertion of
the reporter plasmid construct pDHS45 into flhDC or
'fliA mutant strains was confirmed by Southern analysis
(Fig. 1; data not shown). When bacteria were grown under standard
conditions, -galactosidase activity was reduced to ~20% of
wild-type levels in either YEDS29 (flhDC) or YEDS18
(fliA) compared to YEDS16 (Fig. 3C). Parent strains JB400v
and GY460 do not produce detectable phospholipase activity on plates.
To confirm that the flhDC or 'fliA mutation
was responsible for the decreased -galactosidase production, each
mutation was complemented with the functional gene(s) carried on a
plasmid, pGY10 (50) or pJB222 (2), respectively.
For comparison, the plasmid vector pTM100 was introduced into the
strains as well. The 'fliA mutation was complemented to
wild-type levels of -galactosidase activity by pJB222. Similarly,
the flhDC mutation was complemented by pGY10 yet it
induced even greater -galactosidase activity than that of the wild
type, YEDS16 (Fig. 3C). In neither case did the pTM100 vector alone
complement the mutation. To confirm that the patterns of
transcriptional regulation determined using the reporter fusion
reflected the production of YplA protein, Y. enterocolitica mutant strains with and without complementing plasmids were examined by
Western analysis using antiserum generated against YplA. For all
strains, the amount of YplA correlated with levels of lacZ expression (Fig. 3D). Thus, the results demonstrate that
yplA is part of the flagellar regulon and are consistent
with direct promotion of yplA transcription by FliA
(F).
FliA-dependent regulation would provide a mechanism for both inhibition
of yplA expression by ionic strength and temperature regulation of yplA. The flagellin genes fleABC
are similarly repressed at 37°C and expressed at 26°C, and their
expression is dependent on FliA (F) (23). In
vitro, fliA expression is immediately arrested if the
temperature is increased to 37°C, which explains the loss of motility
and reduced expression of flagellin genes and yplA (24). Repression of yplA expression by ionic
strength may also reflect its regulation as part of the flagellar
regulon. Motility and flagellin production are also repressed by ionic
strength, although the mechanism has not been elucidated
(50). Thus far, characterization of the Y. enterocolitica flagellar regulatory cascade has determined that it
is very similar to the E. coli and Salmonella
paradigm (21, 30). The master regulator flhDC is
required for expression of fliA, and FliA
(F), in turn, promotes expression of the class III
flagellar genes which complete the flagellum. Therefore, FlhDC is
needed for expression of both class II and III genes but FliA is only
necessary for expression of class III genes. Consequently, the effects
of mutations in flhDC and fliA on expression are
consistent with direct promotion of yplA transcription by
F, possibly by binding to the identified
F consensus site. With respect to the patterns of
regulation by oxygen tension (Fnr), catabolite repression (cAMP-Crp),
and a flagellar regulator (FliA), the behavior of yplA is
identical to that of phlA of S. liquefaciens
(16, 17). Indeed, the organization of the Y. enterocolitica yplXAB and S. liquefaciens orfXphlAB loci and the proteins encoded therein are very similar. Since both
Serratia and Yersinia spp. are members of the
family Enterobacteriaceae, it is certainly possible that
other species have phospholipase genes at similar loci. The potential
role of the PhlA phospholipase in pathogenesis has not been addressed,
although S. liquefaciens is considered an opportunistic pathogen.
Based on the E. coli and Salmonella paradigm,
flhDC is predicted to be under catabolite repression
control; cAMP-Crp is required for flhDC transcription
(21, 30). Interestingly, for Y. enterocolitica, the response to the addition of glucose differs for flagellin (and
motility); with or without the addition of glucose, Y. enterocolitica is motile and similar levels of flagellin are
produced (50). However, the phospholipase production appears
to be catabolite repressed. This is consistent with the presence of a
cAMP-Crp site upstream of yplA and suggests one mechanism to
explain why yplA regulation does not always parallel the
flagellar regulon. These data suggest that other regulatory mechanisms
are superimposed on yplA expression, in addition to
regulation by FliA and FlhDC.
Another consideration for the phospholipase is its secretion into the
extracellular milieu, which is undoubtedly necessary for its role as a
virulence factor. Indeed, a number of bacterial phospholipases have
been identified as virulence factors, and without exception, they are
all secreted proteins (reviewed in references 44 and
47). This Y. enterocolitica phospholipase has been shown to utilize the flagellar type III secretion apparatus (49). The expression of class II flagellar genes encoding
this apparatus is dependent on FlhDC but does not require FliA or
expression of the class III genes. Interestingly, the class II
flagellar genes flhBAE of Y. enterocolitica are
not transcriptionally regulated by temperature (14). As FlhA
and FlhB are thought to be part of the secretion apparatus (1,
30), a functional flagellar type III secretion apparatus may be
produced at 37°C although the flagellum is not complete and the
yersiniae are not motile. Production of a functional flagellar
secretion apparatus without a functioning flagellum might appear
wasteful, unless the flagellar secretion apparatus serves another
purpose, such as secretion of nonflagellar proteins.
This work was supported by National Institutes of Health grants
AI27342 and AI01230 to V.L.M., AI09265 to D.H.S., and 5T AI0123 to
G.M.Y.
| 1.
|
Aizawa, S.
1996.
Flagellar assembly in Salmonella typhimurium.
Mol. Microbiol.
19:1-5[CrossRef][Medline].
|
| 2.
|
Badger, J.
1996.
Ph.D. thesis.
University of California, Los Angeles.
|
| 3.
|
Baumler, A. J.,
R. M. Tsolis,
A. W. M. van der Velden,
I. Stojiljkovik,
A. Anic, and F. Heffron.
1996.
Identification of a new iron regulated locus of Salmonella typhi.
Gene
183:207-213[CrossRef][Medline].
|
| 4.
|
Becker, S.,
G. Holinghaus,
T. Gabrielczyk, and G. Unden.
1996.
O2 as the regulatory signal for FNR-dependent gene regulation in Escherichia coli.
J. Bacteriol.
178:4515-4521[Abstract/Free Full Text].
|
| 5.
|
Bercovier, H., and H. H. Mollaret.
1984.
Yersinia, p. 498-506.
In
N. Krieg, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore, Md.
|
| 6.
|
Borodovsky, M.,
K. E. Rudd, and E. V. Koonin.
1994.
Intrinsic and extrinsic approaches for detecting genes in a bacterial genome.
Nucleic Acids Res.
22:4756-4767[Abstract/Free Full Text].
|
| 7.
|
Botsford, J. L., and J. G. Harman.
1992.
Cyclic AMP in prokaryotes.
Microbiol. Rev.
56:100-122[Abstract/Free Full Text].
|
| 8.
|
Bottone, E. J.
1981.
Yersinia enterocolitica.
CRC Press, Inc., Boca Raton, Fla.
|
| 9.
|
Brubaker, R. R.
1991.
Factors promoting acute and chronic disease caused by yersiniae.
Clin. Microbiol. Rev.
4:309-324[Abstract/Free Full Text].
|
| 10.
|
Cornelis, G.,
Y. Laroche,
G. Balligand,
M.-P. Sory, and G. Wauters.
1987.
Y. enterocolitica, a primary model for bacterial invasiveness.
Rev. Infect. Dis.
9:64-87[Medline].
|
| 11.
|
Cornelis, G.,
J.-C. Vanooteghem, and C. Sluiters.
1987.
Transcription of the yop regulon from Y. enterocolitica requires trans-acting pYV and chromosomal genes.
Microb. Pathog.
2:367-379[CrossRef][Medline].
|
| 12.
|
de Koning-Ward, T. F., and R. M. Robins-Browne.
1995.
Contribution of urease to acid tolerance in Yersinia enterocolitica.
Infect. Immun.
63:3790-3795[Abstract].
|
| 13.
|
Delor, I., and G. R. Cornelis.
1992.
Role of Yersinia enterocolitica Yst toxin in experimental infection of young rabbits.
Infect. Immun.
60:4269-4277[Abstract/Free Full Text].
|
| 14.
|
Fauconnier, A.,
A. Allaoui,
A. Campos,
A. Van Elsen,
G. Cornelis, and A. Bollen.
1997.
Flagellar flhA, flhB, and flhE genes, organized in an operon, cluster upstream of the inv locus in Yersinia enterocolitica.
Microbiology
143:3461-3471[Abstract].
|
| 15.
|
Fleischmann, R. D.,
M. D. Adams,
O. White,
R. A. Clayton,
E. F. Kirkness,
A. R. Kerlavage,
C. J. Bult,
J. Tomb,
B. A. Dougherty,
J. M. Merrick,
K. McKenney,
G. G. Sutton,
W. FitzHugh,
C. A. Fields,
J. D. Gocayne,
J. D. Scott,
R. Shirley,
L. I. Liu,
A. Glodek,
J. M. Kelley,
J. F. Weidman,
C. A. Phillips,
T. Spriggs,
E. Hedblom,
M. D. Cotton,
T. Utterback,
M. C. Hanna,
D. T. Nguyen,
D. M. Saudek,
R. C. Brandon,
L. D. Fine,
J. L. Fritchman,
J. L. Fuhrmann,
N. S. Geoghagen,
C. L. Gnehm,
L. A. McDonald,
K. V. Small,
C. M. Fraser,
H. O. Smith, and J. C. Venter.
1995.
Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.
Science
269:496-512[Abstract/Free Full Text].
|
| 16.
|
Givskov, M.,
L. Ebert,
G. Christiansen,
M. J. Benedik, and S. Molin.
1995.
Induction of phospholipase- and flagellar synthesis in Serratia liquefaciens is controlled by expression of the flagellar master operon flhD.
Mol. Microbiol.
15:445-454[Medline].
|
| 17.
|
Givskov, M., and S. Molin.
1992.
Expression of extracellular phospholipase from Serratia liquefaciens is growth-phase-dependent, catabolite-repressed and regulated by anaerobiosis.
Mol. Microbiol.
6:1363-1374[CrossRef][Medline].
|
| 18.
|
Givskov, M., and S. Molin.
1993.
Secretion of Serratia liquefaciens phospholipase from Escherichia coli.
Mol. Microbiol.
8:229-242[CrossRef][Medline].
|
| 19.
|
Givskov, M.,
L. Olsen, and S. Molin.
1988.
Cloning and expression in Escherichia coli of the gene for extracellular phospholipase A1 from Serratia liquefaciens.
J. Bacteriol.
170:5855-5862[Abstract/Free Full Text].
|
| 20.
|
Harlow, E., and D. Lane.
1988.
Antibodies: a laboratory manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 21.
|
Helman, J. D.
1991.
Alternative sigma factors and the regulation of flagellar gene expression.
Mol. Microbiol.
5:2875-2882[Medline].
|
| 22.
|
Iriarte, M.,
J. C. Vanooteghem,
I. Delor,
R. Diaz,
S. Knutton, and G. R. Cornelis.
1993.
The Myf fibrillae of Yersinia enterocolitica.
Mol. Microbiol.
9:507-520[Medline].
|
| 23.
|
Kapatral, V., and S. A. Minnich.
1995.
Co-ordinate, temperature-sensitive regulation of the three Yersinia enterocolitica flagellin genes.
Mol. Microbiol.
17:49-56[Medline].
|
| 24.
|
Kapatral, V.,
J. W. Olson,
J. C. Pepe,
V. L. Miller, and S. A. Minnich.
1996.
Temperature-dependent regulation of Yersinia enterocolitica class III flagellar genes.
Mol. Microbiol.
19:1061-1071[CrossRef][Medline].
|
| 25.
|
Kapperud, G.,
E. Namork,
M. Skurnik, and T. Nesbakken.
1987.
Plasmid-mediated surface fibrillae of Yersinia pseudotuberculosis and Yersinia enterocolitica: relationship to the outer membrane protein YOP1 and possible importance for pathogenesis.
Infect. Immun.
55:2247-2254[Abstract/Free Full Text].
|
| 26.
|
Kinder, S. A.,
J. L. Badger,
G. O. Bryant,
J. C. Pepe, and V. L. Miller.
1993.
Cloning of the YenI restriction endonuclease and methyltransferase from Yersinia enterocolitica serotype O:8 and construction of a transformable RM+ mutant.
Gene
136:271-275[CrossRef][Medline].
|
| 27.
|
Kolter, R.,
M. Inuzuka, and D. R. Helinski.
1978.
Transcomplementation-dependent replication of a low molecular weight origin fragment from plasmid R6K.
Cell
15:1199-1208[CrossRef][Medline].
|
| 28.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[CrossRef][Medline].
|
| 29.
|
Lambert de Rouvroit, C.,
C. Sluiters, and G. R. Cornelis.
1992.
Role of the transcriptional activator, VirF, and temperature in the expression of the pYV plasmid genes of Yersinia enterocolitica.
Mol. Microbiol.
6:395-409[Medline].
|
| 30.
|
MacNab, R. M.
1996.
Flagella and motility, p. 123-145.
In
F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed., vol. 1. ASM Press, Washington, D.C.
|
| 31.
|
Maniatis, T.,
E. F. Fritsch, and J. Sambrook.
1982.
Molecular cloning: a laboratory manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 32.
|
Mekalanos, J. J.
1983.
Duplication and amplification of toxin genes in Vibrio cholerae.
Cell
35:253-263[CrossRef][Medline].
|
| 33.
|
Michiels, T., and G. R. Cornelis.
1991.
Secretion of hybrid proteins by the Yersinia Yop export system.
J. Bacteriol.
173:1677-1685[Abstract/Free Full Text].
|
| 34.
|
Mikulskis, A. V.,
I. Delor,
V. H. Thi, and G. R. Cornelis.
1994.
Regulation of the Yersinia enterocolitica enterotoxin Yst gene. Influence of growth phase, temperature, osmolarity, pH and bacterial host factors.
Mol. Microbiol.
14:905-915[CrossRef][Medline].
|
| 35.
|
Miller, J. H.
1972.
Experiments in molecular genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 36.
|
Pederson, K. J., and D. E. Pierson.
1995.
Ail expression in Yersinia enterocolitica is affected by oxygen tension.
Infect. Immun.
63:4199-4201[Abstract].
|
| 37.
|
Pepe, J. C.,
J. L. Badger, and V. L. Miller.
1994.
Growth phase and low pH affect the thermal regulation of the Yersinia enterocolitica inv gene.
Mol. Microbiol.
11:123-135[Medline].
|
| 38.
|
Pierson, D. E., and S. Falkow.
1993.
The ail gene of Yersinia enterocolitica has a role in the ability of the organism to survive serum killing.
Infect. Immun.
61:1846-1852[Abstract/Free Full Text].
|
| 39.
|
Portnoy, D. A.,
S. L. Moseley, and S. Falkow.
1981.
Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis.
Infect. Immun.
31:775-792[Abstract/Free Full Text].
|
| 40.
|
Saier, M. H. J.,
B. U. Feucht, and M. T. McCaman.
1975.
Regulation of intracellular adenosine cyclic 3':5'-monophosphate levels in Escherichia coli and Salmonella.
J. Biol. Chem.
250:7593-7601[Abstract/Free Full Text].
|
| 41.
|
Schmiel, D. H.,
E. Wagar,
L. Karamanou,
D. Weeks, and V. L. Miller.
1998.
Phospholipase A of Yersinia enterocolitica contributes to pathogenesis in a mouse model.
Infect. Immun.
66:3941-3951[Abstract/Free Full Text].
|
| 42.
|
Simon, R.,
U. Priefer, and A. Puhler.
1983.
A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria.
Bio/Technology
1:784-791[CrossRef].
|
| 43.
|
Sleisenger, M. H.
1981.
Pathophysiology of the gastrointestinal tract.
The W. B. Saunders Co., Philadelphia, Pa.
|
| 44.
|
Songer, J. G.
1997.
Bacterial phospholipases and their role in virulence.
Trends Microbiol.
156:156-161.
|
| 45.
|
Southern, E. M.
1975.
Detection of specific sequences among DNA fragments separated by gel electrophoresis.
J. Mol. Biol.
98:503-517[CrossRef][Medline].
|
| 46.
|
Spiro, S., and J. R. Guest.
1987.
Regulation and over-expression of the fnr gene of Escherichia coli.
J. Gen. Microbiol.
133:3279-3288[Medline].
|
| 47.
|
Titball, R. W.
1998.
Bacterial phospholipases.
J. Appl. Microbiol.
84:127S-137S.
|
| 48.
|
Wagner, A. F.,
M. Frey,
F. A. Neugebauer,
W. Schafer, and J. Knappe.
1992.
The free radical in pyruvate formate-lyase is located on glycine-734.
Proc. Natl. Acad. Sci. USA
89:996-1000[Abstract/Free Full Text].
|
| 49.
|
Young, G. M.,
D. H. Schmiel, and V. L. Miller.
1999.
A new pathway for the secretion of virulence factors by bacteria: the flagellar export apparatus functions as a protein-secretion system.
Proc. Natl. Acad. Sci. USA
96:6456-6461[Abstract/Free Full Text].
|
| 50.
|
Young, G. M.,
M. Smith,
S. A. Minnich, and V. L. Miller.
1999.
The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility.
J. Bacteriol.
181:2823-2833[Abstract/Free Full Text].
|
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Present address: Department of Food Science and Technology,
University of California, Davis, Davis, CA 95616.
