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Applied and Environmental Microbiology, October 2001, p. 4560-4565, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4560-4565.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mutations in the Listerial proB Gene Leading to
Proline Overproduction: Effects on Salt Tolerance and Murine
Infection
Roy D.
Sleator,
Cormac
G. M.
Gahan, and
Colin
Hill*
Department of Microbiology and National Food
Biotechnology Centre, University College Cork, Cork, Ireland
Received 5 March 2001/Accepted 16 July 2001
 |
ABSTRACT |
The observed sensitivity of Listeria monocytogenes to
the toxic proline analogue L-azetidine-2-carboxylic acid
(AZ) suggested that proline synthesis in Listeria may be
regulated by feedback inhibition of 
-glutamyl kinase (GK), the first
enzyme of the proline biosynthesis pathway, encoded by the
proB gene. Taking advantage of the Epicurian
coli mutator strain XL1-Red, we performed random mutagenesis of
the recently described proBA operon and generated three
independent mutations in the listerial proB homologue, leading to proline overproduction and salt tolerance when expressed in
an E. coli (
proBA) background. While each of
the mutations (located within a conserved 26-amino-acid region of GK)
was shown to confer AZ resistance (AZr) on an L. monocytogenes proBA mutant, listerial transformants failed
to exhibit the salt-tolerant phenotype observed in E. coli. Since proline accumulation has previously been linked to the virulence potential of a number of pathogenic bacteria, we analyzed the effect of
proline overproduction on Listeria pathogenesis. However, our results suggest that as previously described for proline
auxotrophy, proline hyperproduction has no apparent impact on the
virulence potential of Listeria.
| |
INTRODUCTION |
Genetic and physiological analysis
of proline accumulation in both prokaryotic and eukaryotic systems
(11, 20) has provided evidence that is consistent with
diverse functions of proline, not only as a source of energy, carbon
and nitrogen but also as an effective osmolyte (1, 10, 11,
23) and more recently as a potential virulence factor for a
number of pathogenic bacteria (2, 12, 33).
While proline can be synthesized from ornithine in both plants and
animals (18), glutamate is the primary precursor for proline biosynthesis in bacteria (23) and in osmotically
stressed plant cells (14). Bacterial proline synthesis
from glutamate occurs via three enzymatic reactions, catalyzed by
-glutamyl kinase (GK) (proB product, EC 2.7.2.11),
-glutamyl phosphate reductase (GPR) (proA product, EC
1.2.1.41), and 1-pyrroline-5-carboxylate reductase (P5C)
(proC product, EC 1.5.1.2). For the majority of bacteria the
proB and proA genes constitute an operon, which
is distant from proC on the chromosome. In plants, e.g.,
Vigna aconitifolia and Arabidopsis, the first two steps of
proline biosynthesis from glutamate are catalyzed by
1-pyrroline-5-carboxylate synthetase (P5CS), a
bifunctional enzyme with both GK and GPR activities at the N- and
C-terminal domains, respectively (18).
For both prokaryotic and eukaryotic systems, proline synthesis from
glutamate is regulated by feedback inhibition of the first enzyme in
the pathway. Studies on purified enzymes suggest that in addition to
proline-mediated inhibition, the -glutamyl kinase activities of GK
and P5CS are also modulated to a lesser extent by glutamate and ADP,
thereby tuning proline synthesis to cellular substrate and energy
availability (37, 39). Proline hyperproducing strains of
bacteria, exhibiting reduced proline-mediated feedback inhibition of GK
activity (a result of single-base-pair substitutions in either the
bacterial proB gene [13, 22, 28, 29, 32] or
the 5' domain of the plant P5CS coding region [39]),
have been isolated based on their resistance to toxic proline analogues (L-azetidine-2-carboxylic acid [AZ]
[15] and 3,4-dehydro-DL-proline, compounds
which inhibit GK activity while not interfering with protein synthesis
[23]).
In addition to the obvious advantages for commercial amino acid
synthesis (29), the osmoprotective properties of proline overproduction (19) have led to the development of
transgenic drought-resistant plants (17). However, since
proline may function as a potential virulence factor (2, 12,
33) and is known to facilitate the growth of certain pathogenic
bacteria at elevated osmolarities (9), the use of
transmissible genetic elements encoding proline hyperproduction may
lead to undesirable consequences, if introduced prematurely into the
natural environment.
Previously we described the isolation and characterization of the
listerial proBA operon (35). In this study we
generated proB mutants which overproduce proline, and we
assess the contribution of such overproduction to the growth and
survival of Listeria monocytogenes, both in hypersaline
environments and during infection of an animal (murine) model.
| |
MATERIALS AND METHODS |
Bacterial strains, plasmids, and culture conditions.
Bacterial strains and plasmids used in this study are listed in Table
1. Escherichia coli strains
were grown at 37°C either in Luria Bertani (LB) medium
(26) or M9 minimal medium (GIBCO/BRL, Eggenstein, Federal
Republic of Germany) containing appropriate additional requirements.
L. monocytogenes strains were grown either in brain heart
infusion broth (Oxoid, Unipath Ltd., Basingstoke, United Kingdom) or in
chemically defined minimal medium (DM) (31). Blood agar
plates consisted of blood agar base (Lab M) to which 5% sheep blood
was added following autoclaving. Where necessary, proline and its
analogues (AZ and 3,4-dehydro-DL-proline) (Sigma Chemical
Co., St. Louis, Mo.) were added to the growth medium at the appropriate
concentration, as filter-sterilized solutions. Antibiotics when needed
were made up as described by Maniatis et al. (26) as
concentrated stocks and added to media at the required levels. Where
indicated, media osmolarity was adjusted by the addition of NaCl.
DNA manipulations and sequence analysis.
Routine DNA
manipulations were performed as described by Maniatis et al.
(26). Plasmid DNA was isolated using the Qiagen QIAprep
Spin Miniprep Kit (Qiagen, Hilden, Federal Republic of Germany).
E. coli was transformed by standard methods
(26), and electrotransformation of L. monocytogenes was achieved by the protocol outlined by Park and
Stewart (30). PCR reagents (Taq polymerase and
deoxynucleoside triphosphates) were purchased from Boehringer GmbH
(Mannheim, Germany) and used according to the manufacturer's
instructions with a Hybaid (Middlesex, United Kingdom) PCR
express system. Oligonucleotide primers for PCR and sequence purposes
were synthesized on an oligo 1000M DNA synthesizer (Beckman
Instruments Inc., Fullerton, California). Nucleotide sequence
determination was performed on an ABI 373 automated sequencer using the
BigDye Terminator sequence kit (Lark Technologies, Inc. Essex, United
Kingdom). Nucleotide and protein sequence analysis were done using
Lasergene (DNASTAR, Ltd., London, United Kingdom). The nucleotide
sequence of the proBA operon in L. monocytogenes can be accessed from the GenBank database (accession no.
AF282880).
Generation of proline analogue-resistant mutants.
The
plasmid pCPL9, harboring the listerial proBA operon, was
transformed into the mutator strain Epicurian coli XL1-Red
(Stratagene), and transformants were selected on LB plates containing
chloramphenicol (30 µg/ml). Transformants were then pooled and grown
overnight at 37°C in LB broth. Randomly mutated plasmid DNA extracted
from this culture was then used to transform the proline synthesis mutant E. coli CSH26. Mutations leading to proline
overproduction were selected by plating transformants on M9 minimal
medium containing 5 mM AZ. These transformants were then pooled and
grown in M9 containing 4% added NaCl, to select for mutations encoding
proline hyperproduction leading to osmotolerance. Plasmids isolated
from the resultant osmotolerant AZr CSH26 clones were then
used to transform L. monocytogenes PSOE (proBA) before screening for proline analogue resistance
(AZr at 10 mM concentrations) and salt tolerance (growth in
DM plus 4% added NaCl).
Analysis of proline production.
Proline hyperproduction was
assayed using a modification of the proline bioassay described by
Kosuge and Hoshino (22). The cell-free supernatant from
overnight cultures of proline-producing strains, in proline-deficient
minimal media, was spotted (in 5-µl volumes) onto M9 plates
containing no added proline and seeded with the E. coli
proline auxotroph CSH26 indicator. Proline overproduction and excretion
was confirmed by subsequent growth of the indicator cells. Quantitative
analysis of the proline in the cell extract of putative proline
overproducers was carried out using a 6300 amino acid analyzer (Beckman
Instruments Ltd., High Wycombe, United Kingdom).
Virulence assays.
Bacterial virulence was determined by
intraperitoneal and peroral inoculation of 8-to 12-week-old BALB/c
mice. Intraperitoneal inoculations were carried out as described
previously (34), using overnight cultures of mutant and
wild-type Listeria (4 × 105 cells),
suspended in 0.2 ml of phosphate-buffered saline. For peroral
inoculations, mutant and wild-type strains suspended in buffered saline
with gelatin were mixed at a ratio of 1:1. Mice were infected with
approximately 2 × 109 cells (total) using a
micropipette tip placed immediately behind the incisors. At 3 days
postinfection mice were euthanized, and listerial numbers were
determined by spread plating homogenized samples onto brain heart
infusion broth (for liver and spleen) and blood agar (for Peyer's
patches and small intestine wall and contents) with and without added
chloramphenicol (10 µg/ml). Maintained resistance to both
chloramphenicol and AZ following passage through the mouse model
confirmed plasmid stability phenotypically.
| |
RESULTS AND DISCUSSION |
Random mutagenesis of the listerial proBA operon.
The observed AZ-mediated inhibition of L. monocytogenes
(Fig. 1) indicated that as with the
majority of systems (both prokaryotic and eukaryotic), listerial
proline biosynthesis from glutamate may be regulated by
proline-dependent feedback inhibition of the GK activity. Mutations
leading to proline analogue resistance (and consequential proline
hyperproduction) have been described for a number of organisms and have
in each case been linked to mutations in GK, leading to a decreased
sensitivity of the enzyme for its allosteric effector proline and its
analogues (13, 22, 28, 29, 32).
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FIG. 1.
Growth of L. monocytogenes LO28 ( ) and
PSOE (pCPL12) ( ) bacteria in DM containing 10 mM AZ as determined
using a spectronic 20D+ spectrophotometer. Growth curves of
Listeria containing plasmids with the other ProB mutations
(pCPL13-16) described in the text were identical to that of PSOE
(pCPL12), but for clarity they are excluded from this graph. Log OD
600nm, log optical density at 600 nm.
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|
In an effort to generate proline-hyperproducing strains of
L. monocytogenes, we used a random mutagenesis strategy to introduce
point mutations into the cloned listerial
proBA operon.
Plasmid
pCPL9 (harboring the listerial
proBA locus) was
transformed into
the
E. coli mutator strain XL1-Red.
Mutations in three of the
primary DNA repair pathways of this strain
result in a mutation
rate which is ~5,000-fold higher than that of
the wild type; hence
pCPL9 replication within XL1-Red led in the
introduction of point
mutations throughout the operon. The randomly
mutated pCPL9 "bank,"
designated pCPL9
mut, was
subsequently transformed into the
E. coli proline auxotroph
CSH26, and transformants were selected on minimal medium containing
5 mM AZ. While no colonies were obtained following a control
transformation
with unmutated pCPL9, transformation efficiencies of 75 CFU/µg
of DNA were achieved from pCPL9
mut, colonies
appearing after 36 h at 37°C. Following overnight growth
at
elevated osmolarities, five AZ
r transformants were chosen
at random for further analysis. Proline
production levels of the five
analogue resistant strains were
tested using the proline bioassay in
combination with amino acid
analysis (Fig.
2A). Complementation of the proline
auxotrophic
indicator strain showed that each clone exhibited proline
overproduction
and excretion compared to the parent containing pCPL9.
Proof that
the observed phenotype was the result of mutations in the
cloned
listerial
proBA operon was obtained by
recomplementation studies,
in which plasmid isolated from each of the
complementing clones
once again conferred AZ
r, not only on
the recipient
E. coli CSH26 strain but also on the
listerial
proline auxotroph PSOE (Fig.
1).
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FIG. 2.
Bioassay for proline overproduction using E. coli CSH26 (proBA) as the indicator strain (as
described in Materials and Methods) and concentrations of proline in
the cell extract (as determined by high-pressure liquid chromatography
analysis of the cell-free supernatant) following transformation of
E. coli CSH26 (A) and Listeria PSOE (B) with
mutated proB genes. In each case unmutated pCPL9 expressed
against a proBA background (CSH26 for panel A and PSOE
for panel B) served as the control. Results of high-pressure liquid
chromatography analysis represent the mean value of three independent
experiments. ND, not detected. Similar results were obtained when the
mutants were cultured at elevated osmolarity and in complex broth in
the presence of exogenous osmolytes.
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|
Sequence analysis of the mutated proBA genes.
Plasmid DNA isolated from the five proline-overproducing CSH26 clones
(pCPL12-16; Table 1) was in each case subjected to sequence analysis
of the cloned listerial proBA operon. Nucleotide sequence
comparisons with the wild-type proBA genes revealed a small
number of base substitutions in the mutated operons (Fig. 3A). Interestingly the base changes, each
of which results in an amino acid substitution within a defined
(26-amino-acid) region of the GK enzyme, map closely to previously
isolated mutations leading to proline overproduction in other genera
(13, 22, 28, 29, 32, 39) (Fig. 3B). This highly conserved
region almost certainly represents an important regulatory domain, most probably the enzyme allosteric binding site. Alternatively,
substitutions in this domain may lead to conformational changes
resulting in a loss of the enzyme's allosteric properties.
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FIG. 3.
(A) Point mutations in the listerial proBA
operon leading to proline overproduction and AZ resistance. (B)
Feedback-resistant mutations in the GKs of L. monocytogenes
(L.m), E. coli (E.c), S. marcescens (S.m), T. thermophilus
(T.t), and V. aconitifolia (V.a).
Residues affected by mutations conferring AZ resistance are in
boldface. Conserved residues are shaded.
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|
In all, three independent mutations leading to an altered GK were
obtained: V121I (pCPL12 and pCPL16), A144V (pCPL13), and
E146K (pCPL14
and pCPL15). In addition, pCPL15 also contains an
A-to-G silent
mutation at nucleotide 390 of the
proB gene, as
well as an
I328V substitution in the GPR protein. Interestingly,
mutations leading
to proline overproduction have been observed
in very similar positions
in other genera, although the actual
residues vary. For example, the
amino acid corresponding to the
listerial V121I mutation is also
altered in both
Serratia marcescens and
Thermus
thermophilus, but in both those cases from A to V
(Fig.
3B). Thus,
a change from valine in the listerial GK is matched
by a change to
valine at the equivalent position in these other
genera. The other
mutations at positions 144 and 146 are also
close to a mutation at a
similar position in
E. coli, illustrating
that this also
functions as an important region in the GK allosteric
site.
Effects of proB mutations on salt tolerance.
The
role of proline as an osmoprotectant was first described by Christian
(7, 8), who in 1955 reported that addition of the amino
acid to media of elevated osmolarity could relieve bacterial growth
inhibition. Based on these observations, Csonka (9)
isolated a proline-overproducing mutant of Salmonella
enterica serovar Typhimurium, exhibiting increased salt tolerance.
The mutation (E. coli ProB D107N [13]) was
located on the E. coli episome, F'128, and could
thus be easily transferred to other enteric bacteria (9,
24).
The role of proline as an effective osmolyte has since been described
for a variety of bacteria, including
Listeria (
3,
4). While each of the three mutations described in the previous
section conferred a similar level of resistance to the proline
analogue
AZ in
E. coli, the ProB V121I mutation conferred the
highest
level of osmotolerance at 4% NaCl relative to the control
strain. The
remaining mutations, while not as osmotolerant as
ProB V121I, still
showed significant increases in growth rate
relative to the control at
elevated osmolarities (data not
shown).
Recently we described the isolation and disruption of the listerial
proBA operon, revealing a significant role for proline
synthesis in contributing to the growth and survival of
L. monocytogenes in environments of elevated osmolarity
(
35). In order to further
assess the importance of proline
synthesis, we analyzed the effect
of overproducing proline on the same
characteristics: osmotolerance
and virulence. We introduced all three
independent
proB mutations
leading to proline overproduction
and analogue resistance into
the
Listeria PSOE
(
proBA) background. While each of the mutated
genes
conferred AZ
r on PSOE, the observed levels of proline
overproduction were found
to be approximately 10-fold lower than those
of
E. coli CSH26
(Fig.
2B).
While this evidence (AZ
r and proline overproduction, albeit
at a reduced level) indicated a physiological consequence of the
introduced mutations, none of the mutants exhibited an osmotolerant
phenotype (data not shown). There are a number of possible explanations
for this phenomenon, the most plausible of which concerns the
extreme
turgor requirement of gram-positive bacteria, which can
be as much as
seven times that of their gram-negative counterparts
(
21).
Maintenance of elevated turgor requires the accumulation
of high
cytoplasmic concentrations of compatible solutes: e.g.,
while 0.5 mM
proline is sufficient to promote maximal growth stimulation
in
E. coli at elevated osmolarities (
9), upwards of 10 mM
proline
is required to facilitate growth of
Listeria at a
similar salt
concentration (
4). While this observed
difference in proline
concentration may well reflect the difference in
turgor requirements
of
Listeria and
E. coli, less
efficient proline transport, coupled
possibly with a more rapid
breakdown of the accumulated proline
against the
Listeria
background, cannot be ruled out. In any case
the levels of proline
overproduction observed, while sufficient
to permit growth of
E. coli at otherwise inhibitory salt concentrations,
seem inadequate
to restore sufficient turgor to PSOE
bacteria.
Thus, increasing the capacity to produce proline alone may not be
enough to confer osmotolerance. In
S. marcessens, maximal
proline production (and consequential osmotolerance) resulted
not only
from mutations in the
proB gene leading to proline
hyperproduction
(
29) but also from an unknown mutation
leading to an increased
production of glutamate (the substrate for GK),
in combination
with mutations in the
putA gene, which result
in a decreased rate
of proline catabolism (
38). The lack
of an observed salt tolerance
phenotype, when the
proB
mutations are transformed into the
Listeria background, thus
may reflect either a limiting concentration of
glutamate (and/or ATP)
or degradation of excess proline by the
listerial PutA equivalent.
Strain-specific effects may also contribute
to the observed drop in
proline production and excretion in
Listeria,
given that the
proB mutations were originally isolated against
an
E. coli background and as such are presumably optimized for
this
environment.
Effects of proline overproduction on the virulence potential of
L. monocytogenes.
In addition to its role as an
osmolyte, which in itself could potentially provide a distinct growth
advantage to Listeria when exposed to the elevated
osmolarity (equivalent to 0.3 M NaCl [6]) of the
gastrointestinal tract, proline has also been suggested to function as
a potential virulence factor in certain pathogenic bacteria (2,
12, 33). Recent evidence suggests that at least in plant cells,
proline may also act as a free radical scavenger, protecting the cells
from the damaging effects of oxidative stress (17). Since
an oxygen-dependent respiratory burst is one of the major mechanisms by
which neutrophils and macrophages kill bacteria (25),
proline hyperproduction may shield Listeria from the
oxidative stress encountered within the macrophage phagosome.
To analyze the effects of proline hyperproduction on the virulence
potential of
L. monocytogenes, the plasmid carrying the
ProB
V121I mutation, which gave rise to the most pronounced osmotolerant
phenotype in
E. coli, was used to transform
L. monocytogenes PSOE.
The resulting strain (ProB
++) was
used to infect BALB/c mice, via the intraperitoneal and
peroral routes.
Similar to results obtained previously for proline
auxotrophy
(
27,
35), proline hyperproduction did not affect
colonization of the upper small intestine, nor did it disrupt
invasion
and spread to the internal organs (Table
2). Thus we
conclude that neither proline
hyperproduction nor inactivation
of proline synthesis has any
measurable effect on
Listeria pathogenesis.
Given that
carnitine is most likely the predominant osmolyte in
animal tissues
(
5), the effects if any of mutating
proBA might
well be masked by carnitine uptake, a hypothesis further evidenced
by
the fact that mutations in OpuC (a carnitine transport system)
result
in a significant reduction in the ability of
Listeria to
colonize the upper small intestine and cause subsequent systemic
infection following peroral inoculation (
36).
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TABLE 2.
Recovery of L. monocytogenes LO28 and the ProB
V121I mutant from the tissues of infected mice 3 days after
intraperitoneal and peroral infection
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ACKNOWLEDGMENTS |
We thank László Csonka (Purdue University) for
providing E. coli CSH26.
This work has been supported by funding from BioResearch Ireland and
the Irish Government under the National Development Plan 2000-2006.
C.G.M.G. is the recipient of a Health Research Board (Ireland)
Post-Doctoral Research Fellowship.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University College Cork, Cork, Ireland. Phone:
353-21-4902397. Fax: 353-21-4903101. E-mail:
c.hill{at}exchsvrs.ucc.ie.
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Applied and Environmental Microbiology, October 2001, p. 4560-4565, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4560-4565.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
