JOURNAL OF BACTERIOLOGY, June 2009, p. 3950–3964
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 12
Development of a mariner-Based Transposon and Identification of
Listeria monocytogenes Determinants, Including the Peptidyl-Prolyl
Isomerase PrsA2, That Contribute to Its Hemolytic Phenotype?
Jason Zemansky,1Benjamin C. Kline,1Joshua J. Woodward,1Jess H. Leber,1†
He ´le `ne Marquis,3and Daniel A. Portnoy1,2*
Department of Molecular and Cellular Biology1and School of Public Health,2University of California, Berkeley,
California 94720-3202, and Department of Microbiology and Immunology, Cornell University,
Ithaca, New York 148533
Received 6 January 2009/Accepted 6 April 2009
Listeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the
intracellular pathogen Listeria monocytogenes. In order to identify factors that control the production, activity,
or secretion of this essential virulence factor, we constructed a Himar1 mariner transposon delivery system and
screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately 200 hypohemo-
lytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular
growth defects. Eight mutants with a phenotype were identified, and they contained insertions in the following
genes: lmo0964 (similar to yjbH), lmo1268 (clpX), lmo1401 (similar to ymdB), lmo1575 (similar to ytqI),
lmo1695 (mprF), lmo1821 (similar to prpC), lmo2219 (prsA2), and lmo2460 (similar to cggR). Some of these
genes are involved in previously unexplored areas of research with L. monocytogenes: the genes yjbH and clpX
regulate the disulfide stress response in Bacillus subtilis, and the prpC phosphatase has been implicated in
virulence in other gram-positive pathogens. Here we demonstrate that prsA2, an extracytoplasmic peptidyl-
prolyl cis/trans isomerase, is critical for virulence and contributes to the folding of LLO and to the activity of
another virulence factor, the broad-range phospholipase C (PC-PLC). Furthermore, although it has been
shown that prsA2 expression is linked to PrfA, the master virulence transcription factor in L. monocytogenes
pathogenesis, we demonstrate that prsA2 is not directly controlled by PrfA. Finally, we show that PrsA2 is
involved in flagellum-based motility, indicating that this factor likely serves a broad physiological role.
Listeria monocytogenes is a gram-positive, facultative intra-
cellular pathogen capable of infecting a broad range of animal
hosts, including humans (84). The cell biology of infection has
been well characterized and is a model for pathogenesis. Upon
internalization into host cells, including macrophages and non-
professional phagocytes, L. monocytogenes organisms are ini-
tially enclosed in a single-membrane vacuole. Bacteria rapidly
lyse this primary vacuole and replicate in the cytosol, exploiting
actin-based motility as a means to move within the cytoplasm
and to spread from cell to cell. Actin-based propulsion of
bacteria from the cytoplasm of one cell into the cytoplasm of a
neighboring cell results in the formation of a double-mem-
brane vacuole or secondary vacuole. Bacteria lyse the second-
ary vacuole, and intracellular growth continues (81, 84).
Central to the virulence of L. monocytogenes is the ability to
lyse the primary and secondary vacuoles in order to gain entry
into the host cytosol. Escape from both types of vacuoles is
primarily mediated by the secretion of the cytolysin listeriolysin
O (LLO) (68). Members of a large family of pore-forming
toxins called the cholesterol-dependent cytolysins, LLO mono-
mers bind cholesterol-containing host membranes. Upon bind-
ing, the monomers oligomerize and the resultant complex in-
serts into the membrane, producing pores up to 30 nm in
diameter (1, 68). Bacteria deficient for LLO production or
activity remain trapped within a phagosome (17, 68) and are
unable to replicate in cells, resulting in a 5-log decrease in
virulence in mice compared to the virulence of wild-type (WT)
bacteria (12, 36, 57).
However, LLO activity must be compartmentalized to the
acidic phagosome. Unrestricted activity can lead to premature
host cell lysis, exposing the bacteria to the inhospitable extra-
cellular environment (22, 33). Mutants incapable of restricting
the activity of LLO to the vacuole have been isolated and are
up to 4 orders of magnitude less virulent in vivo than WT
bacteria (13, 22, 23, 45, 46). LLO is therefore regulated at
Expression of hly, the gene encoding LLO, is controlled by
the L. monocytogenes master virulence transcriptional activator
PrfA (24, 71). In addition to hly, PrfA coordinately regulates
the expression of several other genes necessary for L. mono-
cytogenes pathogenesis, such as the broad-range phospholipase
C (PC-PLC). Although a dramatic change in the expression
profile of bacteria occurs during the transition into the infec-
tious life cycle, only 10 genes (including hly) have been dem-
onstrated to be directly regulated by PrfA (7, 24, 71). An
unexplored possibility, therefore, remains that LLO produc-
tion, activity, or secretion is regulated by other extragenic fac-
Transposon mutagenesis remains one of the most important
* Corresponding author. Mailing address: Department of Molecular
& Cell Biology, 510 Barker Hall no. 3202, University of California,
Berkeley, Berkeley, CA 94720-3202. Phone: (510) 643-3926. Fax: (510)
643-6334. E-mail: firstname.lastname@example.org.
† Present address: Department of Microbiology, University of Chi-
cago, Chicago, IL 60637.
?Published ahead of print on 17 April 2009.
tools in bacterial genetics, facilitating the discovery and explo-
ration of gene function and protein interaction. Given that
transposon mutagenesis has previously proved to be an effec-
tive tool in analyzing hemolysin mutants of L. monocytogenes
(18, 36, 57), we constructed a Himar1 mariner-based transpo-
son and performed a sheep’s blood agar screen for mutants
with a hypohemolytic phenotype. We hypothesized that trans-
poson insertion mutants deficient in the production of LLO
would reveal either novel virulence factors or additional roles
for known factors. To our knowledge, there has never been a
published screen that sought to characterize mutants with hy-
Isolated from the horn fly Haematobia irritans, Himar1 is a
member of the Tc1/mariner superfamily of transposable ele-
ments (27, 40, 55, 62). Himar1-based transposon systems pro-
vide an alternative to the most frequently used system in L.
monocytogenes, a Tn917 derivative (4). Tn917-LTV3 is consid-
erably larger in size (22 kb versus 1.4 kb) and has a relatively
low transposition efficiency, a high rate of delivery vector re-
tention, and a tendency for insertional “hot spots” (4, 6, 19). In
comparison, the Himar1-based transposon system provides
several distinct advantages. Transposition requires no addi-
tional factor other than the cognate transposase, and similar
systems have been shown to be effective in multiple bacterial
species, both gram-negative and gram-positive (40, 64). Addi-
tionally, mariner elements have a low site specificity—the dinu-
cleotide TA—an element common in L. monocytogenes (aver-
age GC content of 39%) (21, 40).
The increased genomic coverage of Himar1 transposon-
based libraries (both within and between genes) has also facil-
itated the ease and resolution of negative-selection screens
(65). These screens have proven to be a remarkably useful tool,
not only in identifying new virulence factors, but also in char-
acterizing which of these factors are necessary during different
stages of an infection (8, 35, 43, 65, 66, 86). Furthermore, these
approaches have assisted in assigning function to previously
uncharacterized virulence factors by mapping their genetic in-
Recently, a new Himar1-based transposon system became
available for use with L. monocytogenes (6). This transposon
does not contain features optimal for its use in a negative-
selection screen. Additionally, there is little control over the
complexity of the library generated or over instances of clones
with multiple transposon insertions. Therefore, we constructed
a new Himar1 system for L. monocytogenes based on a different
strategy of transposon delivery, one that minimizes the poten-
tial for multiple transposition events within a single chromo-
some and that allows control over the complexity of a library.
The new transposon also includes elements that allow us to
take advantage of microarray technologies in order to perform
Among the factors identified in the screen that lead to a
hypohemolytic phenotype was prsA2. A peptidyl-prolyl extra-
cytoplasmic cis/trans isomerase, PrsA2 has previously been
shown to contribute to L. monocytogenes virulence, although
the precise mechanism remains unknown (9, 52, 56). Further-
more, previous work found that prsA2 is upregulated upon
PrfA activation and preceded by a putative PrfA box, suggest-
ing that prsA2 is directly regulated by PrfA (9, 52, 56). Here we
demonstrate that PrsA2 is critical for virulence and contributes
to the secretion and activity of LLO and the activity PC-PLC
but is not under direct PrfA control. Additionally, PrsA2 con-
tributes to flagellum-based motility, an aspect of the bacteri-
um’s life cycle separate from infection. These data suggest that
PrsA2 plays a broader physiological role than previously ap-
MATERIALS AND METHODS
Bacterial strains, growth media, and reagents. The bacterial strains used in
this study are listed in Table 1. All Escherichia coli strains were grown in Luria-
Bertani (LB) medium. All strains of L. monocytogenes were grown in either brain
heart infusion (BHI; Difco, Detroit, MI) medium, LB medium, or LB medium
supplemented with 25 mM glucose-1-phosphate and 0.2% activated charcoal,
with the pH adjusted to 7.3 with 50 mM MOPS (morpholinepropanesulfonic
acid) (LB-G1P) as indicated below. All bacterial stocks were stored at ?80°C in
BHI supplemented with 50% glycerol. Murine L2 fibroblasts were passaged in
Dulbecco modified Eagle medium with high glucose (Gibco/Invitrogen, Carls-
bad, CA) supplemented with 1% sodium pyruvate, 1% L-glutamine, and 10%
fetal bovine serum (GemCell, West Sacramento, CA) at 37°C with 5% CO2. The
following antibiotics were used as indicated at the indicated concentrations:
erythromycin (EM), 2 ?g/ml; lincomycin (LM), 25 ?g/ml; streptomycin, 200
?g/ml; chloramphenicol (CM), 7.5 to 20 ?g/ml; and gentamicin (GM), 10 ?g/ml
(Sigma-Aldrich, St. Louis, MO). All restriction enzymes, T4 DNA ligase, Taq
DNA polymerase, VentR DNA polymerase, and respective buffers were ob-
tained from New England Biolabs (NEB; Beverly, MA).
pJZ037 construction. The plasmids and primers used to construct pJZ037 are
listed in Table 1. The transposon was constructed in pUC19. Using the vector
phiMycoMarT7 (65) as a PCR template, primer pair 112 and 24 and pair 29 and
26 were used to amplify the 5? and 3? ends, respectively, of this transposon, which
included the TA insertion site, the inverted repeat, and the T7 promoter oriented
outward (65). Overhangs included in primers 24 and 29 contained a multiple-
cloning site consisting of SmaI, KpnI, PstI, the trinucleotide AAA, SpeI, and
The transposon backbone was ligated into pUC19 in a three-way ligation to
generate pJZ025 using the PstI sites in primers 24 and 29, a SalI site incorporated
by primer 112, and a HindIII site incorporated by primer 26. Primers 30 and 31
amplified the Tn917 ribosomal methyltransferase gene from pLTV3 (4) and
ligated it into the transposon backbone at the PstI and SpeI sites to generate
The transposase and its promoter were also assembled in pUC19. To increase
the stability of the transposase transcript, the 5? untranslated region (5?UTR) of
hly (lmo0202) was first fused upstream of the hyperactive C9 transposase (39).
Primers 113 and 114 were used to amplify the transposase; primer 113 contained
a 64-bp overhang that included the 51-bp constitutive hyper-Pspac promoter
[Pspac(hy)] (59, 67, 73), and a BamHI site; and primer 114 included a SalI and
a HindIII site. This product was ligated into pUC19 using the BamHI and
HindIII sites, resulting in pJZ026.
The transposon was digested out of pJZ029 with BamHI and ligated into
pJZ026 to generate pJZ032. The inclusion of the 5?UTR of hly upstream of the
transposase, however, prevented plasmid curing. Therefore, this copy of the
transposase was digested out of pJZ032 with EagI and HindIII and replaced with
a copy of the transposase generated by primer 134 and primer 114 (which
removed the 5?UTR), resulting in pJZ039. The transposon and transposase were
digested out of pJZ039 with SalI and ligated into the gram-positive suicide vector
pKSV7 to generate pJZ037 (Fig. 1A) (75).
Generation of libraries. Electrocompetent L. monocytogenes organisms were
prepared as previously described (54), with the exception that vegetable peptone
broth (Remel, Lenexa, KS) was used instead of BHI to increase electroporation
efficiency. Approximately 1 ?g of pJZ037 was used to electroporate each 50-?l
aliquot of electrocompetent cells. Bacteria were recovered in 1 ml of vegetable
peptone broth-0.5 M sucrose and plated over approximately 10 100-mm BHI
EM-LM agar plates. Plates were incubated for 48 h at 30°C (the permissive
temperature) and then replica plated onto BHI EM-LM agar plates and incu-
bated overnight at 41.5°C (the nonpermissive temperature) to cure the plasmid.
Colonies were then counted, scraped, and resuspended in BHI-40% glycerol for
storage at ?80°C.
To test for plasmid retention, 10-fold serial dilutions were prepared from a
small frozen aliquot of the library. Each dilution was plated on a BHI plate
containing EM (the resistance marker carried by the transposon) and on a plate
containing CM (the resistance marker carried by the delivery vector).
VOL. 191, 2009 ROLE OF PrsA2 IN LISTERIA HEMOLYTIC ACTIVITY3951
TABLE 1. Oligonucleotide primers, plasmids, and strains used in this study
Sequence (5?33?) or descriptiona
SmaI, KpnI, PstI
PstI, SpeI, XhoI
Himar1 transposon generated library, strain 10403S
Transposon insertion into lmo0964
Transposon insertion into lmo1268
Transposon insertion into lmo1401
Transposon insertion into lmo1609
Transposon insertion into lmo1695
Transposon insertion into lmo1821
Transposon insertion into lmo2219
Transposon insertion into lmo2460
10403S ?prsA(29-291) tRNAArg::pPL2 with construct 1
10403S ?prsA(29-291) tRNAArg::pPL2 with construct 2
10403S ?prsA(29-291) tRNAArg::pPL2 with construct 3
10403S ?prsA(29-291) tRNAArg::pPL2
10403S ?hly prsA2::Himar1
H. Shen and J. F. Miller; 10
aUnderlining indicates restriction enzyme sites. ?prsA(29-291), the deletion in prsA resulting in the removal of amino acids 29 to 291; p-, 5?-phosphate.
3952ZEMANSKY ET AL.J. BACTERIOL.
Blood plate screen for hypohemolytic mutants. Swabs from frozen libraries
were added to BHI and plated directly onto 1% LB-G1P agar supplemented with
5% defibrinated sheep’s blood (HemoStat Laboratories, Dixon, CA) at a
concentration of 150 to 200 colonies per 100- by 15-mm plate. Plates were
incubated for 48 h at 37°C, and zones of hemolysin were evaluated by eye for
both size and extent of translucency. Potential hits were restreaked on BHI
agar containing EM and LM to confirm the presence of the transposon and
the absence of the plasmid. To confirm the phenotype, both mutant and WT
bacteria were grown overnight at 37°C in BHI with aeration and plated on
blood agar plates in a 1:1 ratio. Plates were scanned using Adobe Photoshop
(Adobe Systems, San Jose, CA).
Identification of transposon insertion sites. Chromosomal DNA of 2-ml over-
night cultures of each mutant were extracted using the MasterPure gram-positive
DNA purification kit (Epicentre, Madison, WI), and the instructions were fol-
lowed with the exception that 10 to 15 units of mutanolysin (Sigma) was used for
1 h at 37°C instead of lysozome. A mixture containing 8 pmol/?l of primers
MSPY5 and MSPY3 in 1? T4 DNA ligase buffer was boiled for 5 min and
allowed to cool to room temperature (79), creating a partially double-stranded
“Y-linker.” A reaction mixture containing the 2 ?g of digested genomic DNA, 2
?l of the Y-linker, and 400 U of T4 DNA ligase was allowed to incubate at 16°C
for 12 h before inactivation of the ligase. Approximately one-third of this reac-
tion mixture was used as a template in an initial PCR to enrich for single-
stranded DNA fragments containing the transposon insertion. A 20-?l total
reaction volume that included a 0.02 mM concentration of the deoxynucleoside
triphosphates (Fermentas, Glen Burnie, MD), 1.5 U Taq (NEB), 1? ThermoPol
buffer, and a 0.1 ?M concentration of primer 121 was first incubated at 95°C for
2 min; incubated at 94°C for 1 min, 61°C for 1 min, and 72°C for 1 min for 20
cycles; and then incubated at 72°C for 7 min. An additional 0.2 mM concentra-
tion of the deoxynucleoside triphosphates, 7.5 U of Taq (and appropriate buffer),
and a 1.5 ?M final concentration of primer 121 and of primer 99 were added, and
the reaction volume increased to 100 ?l (total). This mixture was subjected to the
conditions described above, except that the cycle number was increased from 20
to 25 and the final extension time was increased from 7 min to 10 min. The entire
reaction volume was run out on a 1% agarose gel, visible bands were excised, and
the DNA was purified using the QIAquick gel extraction kit (Qiagen, Valencia,
CA). One hundred nanograms of DNA was submitted to the UC Berkeley DNA
Sequencing Facility with primers 184 and 185 for sequencing.
L2 plaque assays. Plaque assays on murine L2 fibroblasts were performed as
previously described (78). Briefly, the optical densities (ODs) of static, overnight
30°C cultures of L. monocytogenes were normalized, washed three times in
phosphate-buffered saline, and allowed to infect monolayers of L2 cells for 1 h.
Cells were washed and overlaid with 3 ml of 0.7% agar and GM. After 3 days at
37°C, an overlay containing 2 ml of 0.7% agar-GM was added, and ?2.5?
neutral red (Sigma-Aldrich) was added. Monolayers were stained overnight, and
plaque size was evaluated using ImageJ (http://rsbweb.nih.gov/ij/). At least three
wells were used per mutant per experiment, and within each experiment the
average size of each strain was measured as a percentage of the average size of
the WT plaques.
Hemolytic-activity assays. Hemolytic-activity assays were performed as previ-
ously described (57, 73), with some modifications. Briefly, 1 ml of a static,
overnight, 30°C LB medium culture of L. monocytogenes was diluted into 9 ml of
LB medium and grown for 5 h at 37°C with aeration. The OD at 600 nm was
determined. Twofold dilutions of culture supernatants were activated in assay
buffer containing 1? phosphate-buffered saline (Gibco/Invitrogen) and 8.5 ?
10?5M cysteine-HCl (final pH, 5.5; Sigma-Aldrich) at 37°C for 30 min before a
1/10 volume of 5% sheep’s blood was added for another 30 min at 37°C. Purified
LLO was first adjusted to an initial concentration of 2 ?g/ml in assay buffer,
before serial dilutions in assay buffer were made. The assay was completed as
described above except that the 30-min activation step was not performed.
Hemolytic units were defined as the reciprocal of the dilution of culture super-
natant that yielded 50% lysis of sheep red blood cells.
Purification of histidine-tagged LLO from L. monocytogenes. Ten-milliliter
samples of overnight cultures of DP-L3481 (20) and DP-L5633 grown at 37°C in
LB medium with aeration were diluted into 1 liter of LB medium and grown for
8 h at 37°C with aeration. Culture supernatants were obtained by centrifugation
at 6,000 ? g for 15 min and purified by Ni-nitrilotriacetic acid affinity chroma-
tography (Qiagen) according to the manufacturer’s recommendations. Samples
were loaded onto the column using gravity. Columns were washed first with 30 ml
of buffer B (70) and then with 30 ml of buffer B with 150 mM imidazole. Bound
LLO was eluted with 15 ml of buffer B with 500 mM imidazole. The eluted
protein was dialyzed twice in 500 ml of storage buffer (70) at 4°C, at first
overnight and then again for 4 h. Toxin was then mixed with 10% (vol/vol)
glycerol and stored at ?80°C. Prior to analysis, thawed samples were centrifuged
at 13,200 ? g for 10 min at 4°C to remove precipitated protein. Protein concen-
tration was determined by diluting the toxin 1:10 in 6 M guanidine-HCl and 20
mM Na2PO4(pH 6.5) and measuring the absorbance intensity at 280 nm, using
the extinction coefficient obtained by primary sequence analysis with the ExPASy
ProtParam tool (http://us.expasy.org/tools/protparam.html) (89).
CIs and total-CFU assays. Competitive indices (CIs) and total-CFU assays
were performed as previously described (2). A total of 1.0 ? 105CFU/ml of the
mutant or, in the case of the CI, a 1:1 ratio of mutant to WT was intravenously
injected into the tail vein of 9- to 14-week-old female C57/B6 mice (The Jackson
Laboratory, Bar Harbor, ME). To confirm the bacterial load of the injection
mixture, dilutions of the input pool were plated onto BHI-streptomycin plates.
Livers and spleen were harvested 48 h postinfection and homogenized in 10 ml
and 5 ml, respectively, of 0.2% NP-40 (Calbiochem, Darmstadt, Germany). For
the CIs, data were obtained for each mutant from at least seven livers and
spleens as previously described (2). For the total-CFU assay, five age-matched
mice were injected per strain, and the experiment was repeated twice. All animal
work was done in accordance with university regulations.
In-frame deletions and complementation. A WT strain carrying an internal
deletion of prsA2 was constructed as previously described (5). Briefly, primer
FIG. 1. Himar1-based transposon delivery system for L. monocytogenes. (A) The vector pJZ037 contains the following features: the ColE1
origin of replication and a ?-lactamase resistance gene (bla) for propagation in E. coli; a gram-positive, temperature-sensitive origin of replication
(pE194ts ori) and CM resistance gene (cat) for plasmid curing in L. monocytogenes (75); the hyperactive C9 variant of the Himar1 transposase (39)
under the control of the pSpac(hy) promoter (59); and the transposon containing the EM resistance gene (ermAM) (4) for transposon insertion
selection flanked on both sides by T7 promoters oriented outwards for negative-selection screens and the inverted repeats (65). Two multiple-
cloning sites (MCS I and MCS II) flank the EM resistance gene. (B) Locations of 197 insertions mapped onto the L. monocytogenes EGD-e
chromosome. Each line represents a different feature (ORF, intergenic region, or rRNA, etc.). The insertions were obtained from multiple ongoing
screens in our lab searching for different phenotypes. The downward-pointing arrow marks the origin of replication.
VOL. 191, 2009 ROLE OF PrsA2 IN LISTERIA HEMOLYTIC ACTIVITY3953
pairs 229 and 230 and 231 and 232 were used to amplify the regions ?900 bp
upstream and downstream of prsA2, respectively. These fragments were fused
using splice overlap extension PCR and were introduced into the L. monocyto-
genes organisms via allelic exchange using pKSV7 (5, 28). Primers 241, 242, and
243 were each used as the 5? primer with primer 249 to generate the comple-
mentation fragments 1, 2, and 3, respectively (see Fig. 4B). These fragments were
ligated into pPL2 to generate pJZ064, pJZ065, and pJZ066, respectively, and
incorporated into the L. monocytogenes chromosome as previously described
Transductions. Transductions were performed as previously described using
the phage U153 (29). Briefly, 107phage grown on the donor strain were incu-
bated with 108recipient bacteria and then transferred to EM-LM-BHI plates.
Colonies were visible after 48 h of incubation at 37°C.
Isolation of bacterial, secreted proteins and subsequent analyses. Overnight
37°C shaking cultures of L. monocytogenes in LB-G1P were back diluted to an
OD at 600 nm of 0.01 in LB-G1P. Cultures were grown for 8 h at 37°C with
shaking, and ODs were taken every hour. Approximately 1.2 ml of culture was
taken from each sample at 8 h and spun at 13,200 ? g to pellet the bacteria.
Supernatants were removed and stored at ?80°C. In order to normalize for
protein secretion during the entire growth curve, the area under the curve was
derived using KaleidaGraph (Synergy Software, Reading, PA). Each sample was
normalized to the lowest value in a 1.2-ml total volume. Proteins were precipi-
tated with a final concentration of 10% trichloroacetic acid (TCA) (Calbiochem),
washed with ice-cold acetone, and resuspended in 2? sample buffer (Invitrogen)
containing 5% ?-mercaptoethanol. The same fraction of each sample was run
out on a 1.0-mm, 12-well NuPAGE 10% Bis-Tris gel plate (Invitrogen). Gels
were either stained using the Novex colloidal-blue staining kit (Invitrogen) or
subjected to Western blot analysis. Following Western transfer, blots were
probed with a rabbit polyclonal LLO antibody and Alexa-Fluor goat anti-
rabbit immunoglobulin G (Molecular Probes, Eugene, OR) or, additionally
where noted below, with an anti-FLAG M2 monoclonal antibody (Stratagene,
Cedar Creek, TX) and goat anti-mouse IRDye 800CW (LiCor Biosciences,
Lincoln, NE) (67). Blots were visualized using the Odyssey infrared imaging
Assay for egg yolk opacity. Egg yolk agar plates were prepared and assay
conditions were performed as previously described (90).
Assay for motility. Overnight 30°C BHI static cultures of L. monocytogenes
were spotted on semisolid (0.35% [wt/vol] agar) BHI plates and incubated for
24 h and 48 h at 30°C.
Construction of a Himar1 mariner transposon delivery sys-
tem. A Himar1-based transposon delivery system, pJZ037, was
constructed for use in L. monocytogenes (Fig. 1A); the plasmids
and primers used are listed in Table 1. To diminish the occur-
rence of multiple transposon hops within single clones and
clonal populations, problems that are associated with a previ-
ously constructed Himar1 system (6, 90; data not shown), as
well as to add control over the complexity of the library, the
hyperactive C9 variant of the Himar1 transposase (39) was
placed under the control of the constitutive, strong, exogenous
promoter Pspac(hy) (59, 67, 73), and transposition events were
allowed to occur on BHI plates. The transposon contains the
Tn917 ribosomal methyltransferase gene (EM resistance) (4)
flanked by the TA insertion sites, the inverted repeats, and T7
promoters oriented outwards from the vector phiMycoMarT7
in order to facilitate negative-selection screens (65). Both
elements have been ligated into the gram-positive suicide vector
Evaluation of the new Himar1 transposon delivery vector. A
library of approximately 30,000 distinct insertion mutants was
generated using pJZ037. Based on the number of colonies that
were CM resistant (the drug marker carried by the delivery
vector) compared to the number that were EM resistant, ap-
proximately 99% of the clones in the library had transposon
insertions and had lost the delivery plasmid. Twenty randomly
selected clones were subjected to analysis via Southern blot-
ting. All 20 clones examined contained a single transposon
insertion, and each insertion site appeared unique (data not
As an additional measure of library coverage, transposon
insertion mutants isolated from three screens in the lab (two
unrelated to the one described in this report), with focus on
different phenotypes, were mapped onto the assembled ge-
nome of L. monocytogenes EGD-e (Fig. 1B). Each site repre-
sents an insertion into a different feature (open reading frame
[ORF] or intragenic region, etc.). This map includes 197 in-
sertions and covers most of the genome, with only minimal
lengths of sequence lacking insertions. The largest of these
regions covers a length of approximately 160,000 bp, a portion
of the chromosome from lmo401 to lmo0539, perhaps indicat-
ing the presence of many essential genes.
An in vivo negative-selection screen was also performed
similarly to previously described screens (66, 86). Preliminary
analysis of the data revealed that genes within the PrfA regu-
lon were negatively selected for growth in vivo in mice and
guinea pigs (J. Leber, J. D. Sauer, J. Zemansky, and D. A.
Portnoy, unpublished observations; C. Cooke, S. Wong, and A.
Bakardjiev, unpublished observations).
A transposon mutant screen for L. monocytogenes genes in-
volved in the production or activity of LLO. To identify deter-
minants that govern LLO production, activity, or secretion, a
screen for hypohemolytic transposon mutants on sheep’s blood
agar was performed. Approximately 50,000 transposon inser-
tions were screened on 1% LB-G1P agar plates containing 5%
defibrinated sheep’s blood. The inclusion of the LB-G1P in-
creased the expression of PrfA-regulated genes, including hly,
and therefore enhanced visual resolution (61). Mutants were
visually scored for a decrease in the size of and/or the opacity
of the zone of hemolysis, with avoidance of small colonies. Two
hundred fifty-one mutants were initially chosen, and 193 were
confirmed and sequenced. The precise insertion site of the
transposon was determined for 162 of these mutants, 111 of
which were unique. These mutants mapped to a total of 57
unique features, including genes and intergenic regions (Table
2). Multiple hly mutants, representing 14 distinct transposon
insertion sites, were isolated. Of the remaining 31 mutants, the
insertion site was determined to within approximately 200 bp.
Most of these sites overlapped features already identified;
however, six were unique (Table 2). These data suggested that
we were approaching saturation, and no additional screening
Analysis of transposon mutants displaying a hypohemolytic
phenotype. The intracellular life cycle of L. monocytogenes can
be evaluated ex vivo by the capacity of bacteria to form plaques
on mouse L2 monolayers. Defects in any component of this life
cycle—in particular, LLO-mediated escape from the primary
vacuole, escape from the secondary vacuole during cell-to-cell
spread, and failure to compartmentalize LLO activity to the
phagosome—can influence plaque size and shape. In previous
studies, small-plaque mutants of L. monocytogenes invariably
had in vivo defects (78). To identify those mutants isolated
from our initial screen likely to have in vivo relevance, the
average plaque sizes of 51 transposon insertion mutants with
the greatest observed hemolytic defect were evaluated (Table
2). Eight mutants consistently developed smaller plaques than
3954 ZEMANSKY ET AL.J. BACTERIOL.
those of the WT: the lmo0964 (similar to yjbH), lmo1268
(clpX), lmo1401 (similar to ymdB), lmo1575 (similar to ytqI),
lmo1695 (mprF), lmo1821 (similar to prpC), lmo2219 (prsA2),
and lmo2460 (similar to cggR) mutants (Table 2). Of these
genes, only two, lmo1695 and lmo2219, have been character-
ized for L. monocytogenes.
The gene lmo1695 encodes a protein with homology to the
multiple-peptide-resistance factor (MprF) of Staphylococcus
aureus, a membrane protein that catalyzes the transfer of lysine
residues to phosphatidylglycerol and is known to have a major
role in conferring resistance to antimicrobial peptides (80).
This gene was first identified in L. monocytogenes as a compo-
TABLE 2. Unique transposon insertions producing a hypohemolytic phenotype, including those with a precisely mapped site
Strain Transposon insertion(s) Gene namea
size range (%)c
Similar to PTS system, beta-glucoside-specific enzyme IIABC
LacI family transcription regulator
See Table 3
See Table 3
See Table 3
See Table 3
See Table 3
See Table 3
Similar to cysteine synthase
Similar to B. subtilis YacL
Similar to anaerobic ribonucleoside triphosphate reductase
Similar to regulatory proteins (DeoR family)
Similar to oxidoreductase
Similar to transcriptional regulator (LacI family)
Similar to transcriptional regulator (NifA/NtrC family)
Similar to fructokinases
Similar to transcriptional regulator
Similar to B. subtilis YjbH
Similar to phosphotransferase system glucose-specific enzyme IIA
Similar to B. subtilis YfhO
Similar to alpha,alpha-phosphotrehalase
Similar to PTS system, trehalose-specific enzyme IIBC
ATP-dependent protease, ATP-binding subunit
Similar to riboflavin kinase and FAD synthase
Similar to aminopeptidase P
hrcA Heat-inducible transcription repressor
Similar to thioredoxin
prpC Similar to putative phosphoprotein phosphatase
Similar to fibronectin binding proteins
Manganese transport transcriptional regulator
drmSimilar to phosphopentomutase
Similar to transcription regulators (GntR family)
Similar to mannnose-6 phosphate isomerase
Similar to N-acetylmuramoyl-L-alanine amidase and to internalin B
prsA2Similar to posttranslocation molecular chaperone
Similar to arsenate reductase
Similar to peptidyl-prolyl cis/trans isomerase
Similar to B. subtilis CggR hypothetical transcriptional regulator
RNA polymerase factor sigma-54
Similar to inosine-monophosphate dehydrogenase
Bifunctional glutamate-cysteine ligase/glutathione synthetase
Similar to purine-nucleoside phosphorylase
Similar to major cold shock protein
Similar to cell division initiation protein
aAs identified by NCBI annotation of the L. monocytogenes EGD-e genome. PTS, phosphotransferase system; FAD, flavin adenine dinucleotide. Blank spaces
indicate that no specific annotation has been assigned.
bQualitative hemolytic defect visually determined by comparison to an adjacent WT colony on a blood agar plate as follows: -, 75%; --, 50%; ---, 25%; ----, 0% of
the activity of the WT colony.
cRelative to the size of the WT in the L2 fibroblast plaque assay. ND, not done.
dThe approximate site of the transposon is mapped.
VOL. 191, 2009ROLE OF PrsA2 IN LISTERIA HEMOLYTIC ACTIVITY3955
nent of the VirR regulon, and bacteria lacking the response
regulator VirR have a reduced ability to colonize and persist in
the livers and spleens of infected mice (49). L. monocytogenes
mprF mutants are attenuated in virulence in mice and have an
increased susceptibility to certain cationic antimicrobial pep-
tides (80). Although no study has identified a relationship
between LLO secretion and MprF, we eliminated this gene
from further analysis, as its role in virulence has already been
explored (80) and we suspect that the role of mprF on LLO
activity is indirect.
The gene lmo2219 is one of two genes in L. monocytogenes
that encodes a protein with significant homology to the extra-
cytoplasmic lipoprotein chaperone peptidyl-prolyl isomerase
PrsA from Bacillus subtilis. An essential protein in B. subtilis,
PrsA contributes to the folding of proteins following Sec-me-
diated translocation (9, 31, 37, 52, 56). Annotated as prsA2
(compared to prsA1 and lmo1444) (56), lmo2219 was first iden-
tified as a putative virulence factor in a transcriptome analysis
of genes differentially regulated by PrfA; the gene was upregu-
lated under conditions in which PrfA was upregulated, and a
putative PrfA binding site was identified upstream of the gene
(52). PrsA2 transcript levels were also found to be upregulated
during intracellular growth (9), and a proteomics approach
found that PrsA2 secretion levels were increased upon PrfA
activation (56). These studies also found that L. monocytogenes
prsA2 mutants have a plaquing defect (9, 56), an intracellular
growth defect in P388D1 murine macrophage-like cells (9),
and a decreased ability to replicate in the livers and spleens of
infected mice (56). Speculation that PrsA2 contributes to the
folding of extracellular virulence factors remains (9, 56). Given
the possibility that one of these substrates could be LLO, we
continued characterizing this mutant (see below).
Of the remaining mutations leading to plaque defects, none
in L. monocytogenes have been characterized. Based on a
BLAST search of the B. subtilis strain 168 genome (http://www
.ncbi.nlm.nih.gov/), the gene lmo0964 is similar to yjbH (37%
identity, 57% similarity), a gene that confers resistance to
nitrosative stress (63). YjbH also plays a role in regulating the
response to disulfide stress by acting as a negative effector of
the transcriptional regulator Spx (41). Upon disulfide stress,
Spx induces the transcription of genes that maintain thiol-
redox homeostasis (53). Interestingly, mutants with null muta-
tions in clpX and clpP, the genes encoding the ATP-powered
AAA? protease ClpXP, have the same phenotype as yjbH
mutants. A model wherein YjbH facilitates the recognition and
degradation of Spx by ClpXP was proposed (41). Consistent
with these findings, a transposon insertion in lmo1268, the L.
monocytogenes clpX gene, was also identified. This mutant has
a blood plate phenotype and plaque defect similar to those of
the lmo0964 mutant (Fig. 2; Table 3).
The gene lmo1821 codes for a protein similar to the B.
subtilis protein phosphatase PrpC (49% identity, 67% similar-
ity). In both L. monocytogenes and B. subtilis, prpC is directly
upstream of the eukaryotic-type serine/threonine kinase prkC;
the genes are cotranscribed and appear to have opposing phys-
iological roles during stationary-phase growth and biofilm and
spore formation (16, 48). Consistent with its plaque defect,
PrkC homologs have been shown to contribute to the virulence
of Enterococcus faecalis, Streptococcus pneumoniae, Streptococ-
cus agalactiae, and Streptococcus pyogenes (15, 32, 38, 60).
Little is known about the remaining genes. The gene
lmo1401 encodes a protein similar to B. subtilis YmdB (65%
identity, 80% similarity) and contains a putative metallo-phos-
phoesterase domain. The gene lmo1575 encodes a protein
similar to B. subtilis YtqI (55% identity, 72% similarity), a
protein shown to have both oligoribonuclease and pAp-phos-
phatase activity [the conversion of 3?(2?)phosphoadenosine 5?
phosphate to AMP] (51). The gene lmo2460 encodes a protein
similar to B. subtilis CggR (54% identity, 75% similarity), a
repressor of the gapA operon, which contains many of the
genes necessary for glycolysis (14, 47).
We continued to characterize six mutants: the lmo0964 (sim-
ilar to yjbH), clpX, lmo1401 (similar to ymdB), lmo1575 (sim-
ilar to ytqI), lmo1821 (similar to prpC), and prsA2 mutants (Fig.
2; Table 3). As with the mprF mutant, the lmo2460 (cggR)
mutant was not examined further as it had a small but repro-
ducible growth defect in broth (G1P medium), making it po-
tentially difficult to separate a specific defect in LLO produc-
tion or activity from a general metabolic defect. The remaining
mutants grew in the same way as the WT (data not shown).
FIG. 2. Blood plate phenotypes for hypohemolytic mutants. Wild-
type (10403S) bacteria were plated at a 1:1 ratio with the following
transposon insertion mutants on LB-G1P agar containing 5% defi-
brinated sheep’s blood: the lmo0964 (similar to yjbH) mutant (A),
lmo1268 (clpX) mutant (B), lmo1401 (similar to ymdb) mutant (C),
lmo1575 (similar to ytqI) mutant (D), lmo1821 (similar to prpC) mu-
tant (E), and lmo2219 (prsA2) mutant (F). Arrows denote mutant
TABLE 3. Plaque sizes of transposon insertion mutants
(% of WT size)a
51.6 ? 5.3***
61.3 ? 2.7***
35.1 ? 4.5*
64.7 ? 5.3**
72.4 ? 3.2***
16.2 ? 1.3***
aMean plaque sizes of murine L2 fibroblasts infected with transposon inser-
tion mutants ? standard deviations, relative to that of the WT strain (the mean
plaque size of the WT strain within each experiment was defined as 100%). Each
mutant strain was analyzed in at least three experiments. To test the hypothesis
that each mean mutant plaque size differs from 100%, a one-sample two-sided t
test was performed using R software (http://www.r-project.org/). ?, P ? 0.005; ??,
P ? 0.001; ???, P ? 0.0005.
3956ZEMANSKY ET AL. J. BACTERIOL.
That each mutant’s phenotype was due to the transposon in-
sertion was confirmed by transduction (29) into a WT back-
ground followed by comparison of the blood plate phenotypes
and plaque defects (data not shown).
To assess defects in either the amount of LLO secreted or
the activity of the secreted toxin, the hemolytic activity present
in culture supernatants of each mutant was determined. As
shown in Table 4, the majority of our mutants have decreased
hemolytic activity. The lmo1575 insertion mutant had the
greatest defect, with an average of 29.6% of the activity of the
WT, followed by mutants with transposon insertions in
lmo0964 (40.3%) and lmo1268 (clpX) (55.9%), an in-frame
deletion of prsA2 (see below) (57.7%), and a transposon in-
sertion in lmo1401 (69.9%). Interestingly, the lmo1821 (prpC)
transposon insertion mutant had essentially the same level of
hemolytic activity as the WT, indicating that the conditions on
the blood agar plate (incubation on solid medium for 48 h)
elicited a phenotype different from that after growth in broth
culture over 5 h.
Western blotting was also performed to assess LLO secre-
tion levels. Although there were small but reproducible qual-
itative differences in the amounts of LLO secreted by the
mutants compared to that secreted by the WT, these differ-
ences were not significantly robust to draw any definitive con-
clusions (data not shown). It is therefore unclear whether the
differences identified in the hemolytic-activity assay are due to
subtle secretion defects or due to the activity of the LLO
secreted (see Discussion).
In vivo analysis of the plaque mutants. To assess the poten-
tial virulence defect of each of our six transposon insertion
mutants in vivo, a CI assay (2) was utilized to quantify the
potential relative replication defect each mutant has in the
livers and spleens of C57BL/6 mice coinfected with WT L.
monocytogenes. For each transposon mutant, five mice were
coinfected with 1.0 ? 105bacteria in an ?1:1 ratio of mutant
to WT via intravenous tail vein injection. Livers and spleens
were harvested 48 h postinfection, and the ratio of WT to
mutant bacteria was assessed (2). The prsA2 insertion mutant
displayed the greatest phenotype, as it was undetectable in our
organ homogenates after several hundred colonies were
patched, consistent with the previously described virulence de-
fect of this mutant of being greater than 2 logs in the liver and
spleen (56). The lmo1401 (ymdB) insertion mutant also dis-
played a significant decrease in bacterial numbers in both the
liver and spleen, with median defect rates of 2.0 and 1.1 logs,
respectively. Consistent with previous studies of the role of
prpC/prkC in virulence in other gram-positive pathogens (15,
32, 38, 60), the transposon insertion in lmo1821 (prpC) had a
median defect rate of 2.2 logs in the liver and a median defect
rate of 1.1 logs in the spleen. Finally, consistent with our
hypothesis that transposon insertions in lmo0964 (yjbH) and
clpX affect the same pathway, the relative decreases in bacte-
rial loads between these two mutants (median defect rates of
1.7 versus 1.6 logs in the liver and 0.6 versus 0.7 log in the
spleen) were similar (Fig. 3).
In-frame deletion and complementation of PrsA2. Given
that the insertion mutation in prsA2 had the greatest defect in
vivo, combined with data (Fig. 2; Table 4) suggesting a link
between PrsA2 and LLO secretion and/or activity, we contin-
ued to characterize this gene. Two different transposon inser-
tions into prsA2 were isolated, one 317 bp from the start site
and one 410 bp from this site (Fig. 4A). Although one group of
investigators was unable to delete PrsA2 (52), this result—that
prsA2 is dispensable—is consistent with the results of two ad-
ditional groups who were able to inactivate this gene either by
making an in-frame deletion (9) or by inserting a plasmid (56).
Both mutants with transposon insertions in prsA2 had the
same blood plate phenotype and plaque defect (data not
shown). For the remainder of this study, the insertion 317 bp
downstream from the start of the gene was used. However, to
confirm that the observed phenotypes were the result of a
disruption of prsA2 and not due to an unlinked mutation, an
in-frame deletion of prsA2, resulting in a removal of amino
acids 9 to 291 (the protein is 293 amino acids long), was carried
out (5). The resulting strain, the ?prsA2 mutant, has the same
plaque defect as that of the strains with either transposon
insertion (Table 5).
FIG. 3. In vivo defects of transposon insertion mutants as mea-
sured by CI. Bacteria were harvested 48 h postinfection from at least
seven mice per mutant, and the ratios of mutant to WT bacteria were
determined for the liver (E) and spleen (‚). All median values are
represented by horizontal lines. We performed one-sample Wilcoxon
tests on the ratios, using R software (http://www.r-project.org/). Under
the null hypothesis of no differences, the mean parameter is assumed
to be one. A two-tailed P value is reported for each mutant. ?, P ?
0.05; ??, P ? 0.005.
TABLE 4. Hemolytic activities of culture supernatants and
Strain or purified protein
lmo0964::Himar1 strain................................................. 40.3 ? 10.6**
lmo1268::Himar1 strain................................................. 59.7 ? 17.1*
lmo1401::Himar1 strain................................................. 69.9 ? 3.9**
lmo1575::Himar1 strain................................................. 29.6 ? 2.6***
lmo1821::Himar1 strain.................................................101.5 ? 14.3
?prsA2 mutant.................................................................... 58.0 ? 13.3*
?prsA2 mutant ? construct 2........................................... 88.7 ? 10.8
Purified protein prsA2::Himar1 LLOHis6......................... 67.2 ? 6.1**
aMean percentages of the hemolytic activities of either culture supernatants
or purified protein ? standard deviations, relative to WT activity, from at least
four experiments. Statistical analysis was performed as described for Table 3. ?,
P ? 0.05; ??, P ? 0.005; ???, P ? 0.0005.
VOL. 191, 2009 ROLE OF PrsA2 IN LISTERIA HEMOLYTIC ACTIVITY3957
To evaluate the in vivo role of prsA2, mice were intrave-
nously injected with WT or ?prsA2 L. monocytogenes and livers
and spleens were harvested 48 h postinfection to determine
total bacterial loads. Consistent with the results of the CI assay
for the transposon insertion into prsA2, the ?prsA2 strain was
greater than 5 logs less virulent in the liver and 2.8 logs less
virulent in the spleen (Fig. 4B). To further confirm the in vivo
role of prsA2, we complemented our in-frame deletion of prsA2
with a chromosomal copy. Previous studies had found an in-
crease in prsA2 transcript and protein levels upon prfA induc-
tion, and a putative PrfA box had been identified 206 bp
upstream of the start of the prsA2 ORF (9, 52, 56). However,
this location places the start of the promoter 90 bp within the
3? end of the upstream gene lmo2218. Of the 10 genes con-
firmed to be directly regulated by PrfA, none has the promoter
located within an upstream gene (71).
To test whether the putative PrfA box was dispensable, three
different constructs to complement the ?prsA2 mutant were
designed, each differing at its 5? end from the others. The first
contains the putative PrfA box (construct 1), the second con-
struct contains just the region directly downstream of the 3?
end of the lmo2218 ORF, and a third control construct con-
tains just the ORF of prsA2 (Fig. 4A). The 3? end of each
construct includes the 131-bp region downstream of prsA2,
including the terminator sequence (Fig. 4A). Each construct
was placed into the integration vector pPL2 (42) and inte-
grated into the chromosome of the ?prsA2 strain. As an addi-
tional control, an empty pPL2 vector was also integrated into
the parent ?prsA2 strain.
The ability of these constructs to complement the plaque
defect of the prsA2 transposon insertion mutant was analyzed.
As expected, both the ?prsA2 strain containing just the ORF
and the ?prsA2 strain with the empty pPL2 vector produced
plaques of the same size as those of the transposon insertion
mutant and the ?prsA2 strain (Table 5). Interestingly, both
construct 1 and construct 2 complemented the transposon mu-
tant equally well, suggesting that the PrfA box was dispensable.
The ability of the strain containing construct 2 to complement
the in-frame deletion in vivo was tested. This construct re-
stored full virulence, as well as complemented the hemolytic
activity defect of the ?prsA2 culture supernatants (Table 4),
again suggesting that the PrfA box is dispensable for prsA2
expression and activity (Fig. 4B).
Possible role for PrsA2 in folding exported LLO. Western
blot analysis of LLO secretion in the ?prsA2 mutants revealed
an unusual banding pattern: in addition to a major band at
approximately 58 kDa (LLO), there were two minor bands at
approximately 43 and 41 kDa (Fig. 5A). This laddering pattern
is absent in the ?prsA2 strain complemented with construct 2.
To confirm that these additional bands were LLO, the trans-
poson insertion mutant was transduced into two different back-
FIG. 4. Virulence of L. monocytogenes prsA2 mutants. (A) Schematic of the prsA2 genomic loci and the three different constructs used to
complement ?prsA2. Each construct differed at its 5? end from the others; construct 1 (#1) contained the identified PrfA box, construct 2 (#2)
contained the region immediately downstream of lmo2218, and construct 3 (#3) contained just the ORF of prsA2. The two triangles below prsA2
indicate the two transposon mutants isolated in this screen. Stem-loop structures denote transcription terminators. (B) WT, ?prsA2, and ?prsA2
strains complemented with construct 2 were analyzed for total bacterial load in the livers (E) and spleens (‚) of mice 48 h postinfection. All median
values are represented by horizontal lines. Each experiment was repeated. We performed paired Student’s t tests on the total bacterial load data,
using R. Under the null hypothesis of no differences, the mean parameter is assumed to be zero. A two-tailed P value is reported for each mutant.
??, P ? 0.005.
TABLE 5. Complementation data
Complemented strainPlaque size (%)a
prsA2::Himar1 strain ? construct 1
prsA2::Himar1 strain ? construct 2
prsA2::Himar1 strain ? construct 3
prsA2::Himar1 strain ? empty pPL2
18.2 ? 4.6*
100.6 ? 5.1
99.1 ? 2.1
16.3 ? 3.2
17.0 ? 3.4
aMean plaque sizes of murine L2 fibroblasts infected with either the strain
with the in-frame deletion of prsA2 or the prsA2 transposon insertion mutant
complemented with different constructs, relative to the WT plaque size, ?
standard deviations. Statistical analysis was performed as described for Table 3.
?, P ? 0.005.
3958 ZEMANSKY ET AL. J. BACTERIOL.
grounds: a WT strain containing an in-frame deletion of hly
(DP-L2161) and a WT strain containing a chromosomal copy
of hly with the point mutation resulting in S44A (DP-L4057),
which relieves the translational inhibition of LLO (LLOS44A).
This strain produces and secretes LLO at a constant rate
throughout the entire growth cycle of the bacteria (29, 34, 69).
These additional strains were subjected to Western blot anal-
ysis. In addition to the lack of the major LLO band in the ?hly
prsA2::Himar1 strain, both lower-molecular-weight bands were
absent, consistent with the hypothesis that these bands were
species of LLO. Additionally, in the LLOS44AprsA2::Himar1
mutant, there was an increase in the size and intensity of the
major LLO band as well as in the smaller bands. Furthermore,
a smaller set of two bands was visible at approximately 33.5 and
32 kDa; further comparison revealed the presence of these
bands in the ?prsA2 mutant (Fig. 5A). These results strongly
suggested that the additional bands in the prsA2 mutant back-
grounds were lower-molecular-mass species of LLO.
We hypothesized that these additional bands might be deg-
radation products. Bacteria have several mechanisms to refold
or degrade misfolded secreted proteins. B. subtilis, for exam-
ple, has several known extracytoplasmic quality control sys-
tems, including the serine protease family members HtrA and
CWBP52 (82). To investigate this possibility further, a C-ter-
minally epitope-tagged copy of LLO was first employed; LLO
contains a cleavable signal sequence at its N terminus (68).
The transposon insertion into prsA2 was transduced into a
WT strain containing a chromosomal copy of LLO with a
C-terminal FLAG tag (LLOFLAG) (DP-L4361). The Western
blot analysis was then repeated on the LLOFLAGand the
prsA2::Himar1 LLOFLAGstrains, with probing for both LLO
and the FLAG epitope. Consistent with our previous results,
when the strain was probed with the anti-LLO antibody, the
discrete, smaller bands were of greater intensity in the
LLOFLAGprsA2 transposon mutant. However, these bands did
not appear when the strain was probed with the anti-FLAG
antibody (Fig. 5B). These results are consistent with our hy-
pothesis that LLO was undergoing C-terminal cleavage in the
Purified LLO secreted by the prsA2 mutant is less active.
Both the WT and ?prsA2 mutant secreted approximately the
same level of full-length LLO, as indicated by Western blot
analysis of proteins isolated from culture supernatants (Fig.
5A). We wondered then if the decrease in LLO hemolytic
activity of the ?prsA2 mutant (Table 4) was due to misfolded
full-length LLO. Alternatively, it is possible that the smaller
fragments of LLO act in a dominant negative fashion, perhaps
by oligomerizing with the full-length toxin but preventing
membrane insertion. To address this possibility, full-length
LLO was purified from WT or prsA2::Himar1 supernatants and
tested for hemolytic activity.
The prsA2 transposon insertion was transduced into a WT
strain of L. monocytogenes containing a C-terminally six-histidine-
tagged allele of LLO (LLOHis6) (DP-L3481) (20). LLOHis6was
isolated from 8-h culture supernatants from both the WT and
prsA2::Himar1 strains by nickel affinity chromatography. The
purity of the eluted toxin was confirmed by analysis of the
preparation with sodium dodecyl sulfate (SDS)-polyacryl-
amide gel electrophoresis (PAGE). Additionally, Western blot
analysis was used to confirm that the smaller N-terminal frag-
ments of LLO (Fig. 4A) were not present (data not shown).
Toxin purified from both the WT and prsA2::Himar1 strains
was then diluted appropriately and analyzed for hemolytic
activity. The activity of purified, full-length LLOHis6isolated
from the prsA2::Himar1 strain was 67.2% of the WT’s activity,
similar to the value obtained for the hemolytic activity ob-
tained the from the ?prsA2 culture supernatants (Table 4).
FIG. 5. PrsA2 affects the secretion of LLO. (A) Western blot of secreted proteins probed with a polyclonal anti-LLO antibody from the
following bacterial strains: the WT (lane 1), ?prsA2 mutant (lane 2), ?prsA2 tRNAArg::pPL2 mutant with construct 2 (lane 3), ?hly prsA2::Himar1
mutant (lane 4), LLOS44Amutant (lane 5), LLOS44AprsA2::Himar1 mutant (lane 6), LLOFLAGstrain (lane 7), and LLOFLAGprsA2::Himar1 strain
(lane 8). The black arrows indicate the increased presence of the two bands at ?42 kDa and at ?33 kDa in the prsA2 mutant backgrounds relative
to bands in the nonmutated or complemented prsA2 background. (B) Western blot analysis of the LLOFLAG(lane 1) and LLOFLAGprsA2::Himar1
(lane 2) strains probed with anti-LLO (red) and anti-FLAG (green). The two probes overlap at the major band at 58 kDa but not at ?42 kDa.
(C) Colloidal Coomassie blue stain of an SDS-PAGE gel of purified secreted proteins from the same strains as those discussed in panel A. The
black arrows indicate the presence of additional bands in the ?prsA2 mutant backgrounds (including the ?hly background), as well as molecular
masses greater than that of LLO.
VOL. 191, 2009 ROLE OF PrsA2 IN LISTERIA HEMOLYTIC ACTIVITY3959
These data are consistent with our hypothesis that PrsA2 is
specifically involved in the proper folding of secreted LLO.
Other potential substrates for PrsA2. Examination of a col-
loidal Coomassie blue-stained SDS-PAGE gel of proteins pre-
cipitated from the supernatants of the multiple prsA2 strains
revealed the presence of several bands, both higher in molec-
ular weight than LLO and lower. These bands are not present
in either the WT or ?prsA2 complemented strains (Fig. 5C).
L. monocytogenes secretes PC-PLC, which contributes to the
ability of the bacteria to escape from the primary and second-
ary vacuoles. The lecithinase activity of PC-PLC, encoded by
plcB, can be assessed by measuring zones of opacity on an LB
agar plate containing egg yolk agar (50, 74, 83). To investigate
whether PC-PLC was affected in the prsA2 mutant, the ?prsA2
mutants were tested for lecithinase activity. The ?prsA2 strain
showed a decreased zone of opacity on egg yolk agar compared
to those of both the WT and construct 2-complemented
?prsA2 strains (Fig. 6A). To increase the resolution of this
assay, the SLCC-5764 (DP-L861) strain of L. monocytogenes
was utilized. SLCC-5764 contains a prfA* allele, a dysregulated
allele of prfA that increases the expression of the PrfA-regu-
lated genes, including plcB (26, 44). The prsA2 transposon
insertion was transduced into this strain, which was spotted on
an egg yolk agar plate with the parent SLCC-5764 strain.
Again, the disruption of prsA2 caused a visible decrease in the
amount of visible lecithinase activity (Fig. 6A).
Given that prfA appears to be dispensable for prsA2 expres-
sion, we investigated whether the disruption of prsA2 causes
the disruption of other exported proteins not involved in vir-
ulence. L. monocytogenes is motile and flagellated at temper-
atures of 30°C and below but nonmotile at 37°C (24, 25). Given
that components of the flagellar machinery are recognized by
elements of the immune system, it has been proposed that L.
monocytogenes actively downregulates the expression of these
components upon infection (25). Therefore, to assess the effect
of PrsA2 on secretion in a context where PrfA activity is low,
the flagellum-based motilities of the mutants were compared.
Cultures of the WT, the ?prsA2 mutant, the ?prsA2 strain
complemented with construct 2, the ?flaA mutant (DP-L4650),
and a strain containing in an in-frame deletion of prfA (DP-
L4317) were all spotted on low-percentage-motility LB agar
plates for 24 and 48 h at 30°C. The ?prsA2 strain had a smaller
swarm pattern than did the WT or the strain complemented
with construct 2 (roughly half the size) (Fig. 6B). This defect
was, however, considerably smaller than the defect in the ?flaA
strain, indicating only a partial loss of swarming ability. This
phenotype is consistent with the previous results from the
blood and egg yolk agar plates: in all instances, there was only
partial loss of activity rather than a complete abrogation. Fi-
nally, the ?prfA strain’s swarm size was equivalent to that of
both the WT and the ?prsA2 strain complemented with con-
struct 2, consistent with the complementation results that PrfA
appears dispensable for prsA2 expression and activity.
In this study, we have described the construction of a
Himar1-based transposon system for use in L. monocytogenes.
This construction decreases many of the issues observed using
previous transposon systems in L. monocytogenes, specifically,
in instances of multiple transposon hops within a single bacte-
rium, clonal populations within a given library (6), and inser-
tion “hot spots” (4). Furthermore, the inclusion of T7 promot-
ers makes negative-selection screens possible. This transposon
delivery system is also effective in Bacillus anthracis (J. Beaber,
J. Zemansky, D. A. Portnoy, R. Calendar, unpublished re-
In order to identify extragenic factors involved in LLO pro-
duction, activity, or secretion, 50,000 transposon insertions
were screened on sheep’s blood agar plates for hypohemolytic
phenotypes. Critical for virulence, LLO is subject to multiple
levels of regulation (68). Given the increased complexity of
Himar1 mariner transposon mutant libraries, the likelihood of
identifying extragenic regulators of LLO, and therefore likely
virulence determinants, was increased. Additionally, to our
knowledge, this study represents the first published screen for
mutants with a hypohemolytic phenotype rather than for those
with an ahemolytic phenotype. As a result of the screen, 193
mutants were initially identified as having a hypohemolytic
phenotype by visual inspection (Table 2). Plaque assays of
murine cells were performed on the 51 mutants with the most
discernible visible defect (Table 2).
Given the importance of hly in virulence, we expected that a
greater fraction of our mutants would exhibit defects escaping
from the primary and secondary vacuoles in the ex vivo plaque
assay (68). Curiously, of the mutants analyzed, only eight had
FIG. 6. PrsA2 affects the secretion of additional substrates. (A) PrsA2 mutants affect PC-PLC (lecithinase activity). Overnight cultures grown
at 30°C were spotted on 5% (wt/vol) egg yolk LB-G1P plates and grown for 24 h. (a) WT; (b) ?plcB mutant (DP-L1935); (c) ?prsA2 mutant; (d)
prsA2::Himar1 mutant; (e) ?prsA2 tRNAArg::pPL2 mutant with construct 1; (f) ?prsA2 tRNAArg::pPL2 mutant with construct 2; (g) SLCC-5764
strain of L. monocytogenes; (h) SLCC-5764 (prsA2::Himar1). (B) PrsA2 mutants affect swarming ability. Overnight cultures grown at 30°C were
spotted on 0.35% BHI plates and grown for 24 h (a to e) or 48 h (f to j) at 30°C. (a and f) WT; (b and g) ?flaA mutant (DP-L4650); (c and h)
?prsA2 mutant; (d and i) ?prsA2 tRNAArg::pPL2 mutant with construct 2; (e and j) ?prfA mutant.
3960 ZEMANSKY ET AL.J. BACTERIOL.
repeatable, substantive plaque defects. One possibility for the
discrepancy between blood plate phenotype and the lack of an
in vivo defect may be that the phenotypes revealed on blood
agar might be too subtle to be physiologically relevant. The
blood plate assay is very sensitive, as zones of hemolysis are the
result of discrete foci of toxin activity occurring over the course
of 48 h.
The screen successfully identified both potential novel viru-
lence factors and additional roles for previously described fac-
tors; in the eight mutants with both a blood plate phenotype
and a plaque defect, six genes—yjbH, clpX, ymdB, ytqI, prpC,
and cggR—have not previously been characterized for L.
monocytogenes. Although the other two genes, mprF and prsA2,
are known to contribute to virulence, a potential relationship
between each of these factors and the secretion of LLO has not
previously been established (9, 52, 56, 80).
The six characterized mutants displayed a range of defects in
the ex vivo plaque assay, the hemolytic-activity assay, and in
vivo (Tables 3 and 4; Fig. 3 and 4). The genes ymdB (the gene
containing the putative metallo-phosphoesterase domain) and
ytqI (encoding the putative oligoribonuclease) have not been
well characterized, and speculating about a potential role in
LLO regulation is difficult. Additional work is necessary in
order to determine whether the phenotypes of these mutants
are the result of a direct relationship with LLO or the PrfA
regulon or a more general pleiotropic effect. However, the
identification of these genes does suggest that there are as-yet-
unidentified factors or pathways that regulate LLO production,
secretion, or activity.
More intriguing are the transposon insertions in yjbH, clpX,
prpC, and prsA2. The association of a strain with a mutation in
prpC, the phosphatase directly upstream of the eukaryotic-
kinase-type serine/threonine kinase, with a virulence defect
parallels a similar defect in prpC prkC mutants of other gram-
positive pathogens (15, 32, 38, 60). Based on BLAST align-
ments (http://blast.ncbi.nlm.nih.gov/), PrpC is approximately
40% identical and 60% similar to its likely homolog in each of
S. pyogenes M1 group A streptococci, S. agalactiae group B
streptococci, E. faecalis, and S. pneumoniae. Equally intriguing
is a recent study that has suggested a role for PrkC in B. subtilis
in sensing the extracellular environment by binding fragments
of peptidoglycan (72). It is therefore tempting to ascribe a
direct role for prpC and prkC in L. monocytogenes virulence,
possibly by allowing the bacteria to “sense” its environment.
However, while mutations in these genes are known to cause
changes in the expression of certain virulence factors, they
have also been linked to effects on growth, morphology, and
cell division and other pleiotropic effects (32, 60). Additional
work is required to discern the precise relationship between
this kinase/phosphatase pair and members of the PrfA regulon.
The reason a difference exists between the phenotype on the
blood agar plate (Fig. 2) and the results of the hemolytic-
activity assay (Table 4) remains unclear.
yjbH and clpX mutants displayed similar blood plate pheno-
types, similar hemolytic activities, and similar virulence defects
(Fig. 2 and 3; Tables 3 and 4). In B. subtilis, their predicted
homologs have recently been characterized as regulating the
activity of the disulfide stress regulator Spx (41). This tran-
scription factor is responsible for maintaining thiol-redox ho-
meostasis (41, 53), and it is tempting to speculate that the L.
monocytogenes spx (lmo2191) gene product may answer one of
the long-standing questions regarding the toxin. The choles-
terol-dependent cytolysins, including LLO, are also known as
the “thiol-activated” cytolysins due to the presence of a con-
served cysteine residue (76). Crude preparations of these tox-
ins are readily oxidized and require the addition of a reducing
agent to reverse the effects. However, this effect diminishes as
the purity of the preparation increases, and mutating the cys-
teine to an alanine does not eliminate LLO hemolytic activity
(58). One possible explanation for the conservation of this
cysteine residue is that it serves a regulatory role and may be
the target site of some external molecule. Perhaps L. monocy-
togenes Spx controls a factor that binds to this residue, seques-
tering activity. YjbH and/or ClpX would then work to limit the
repression by Spx. Consistent with this hypothesis, a transpo-
son insertion into a gene encoding thioredoxin-like protein
(lmo1609) was identified (Table 2), although this mutation did
not lead to a significant plaque defect.
Most interesting was our finding that a transposon insertion
into prsA2 affected LLO secretion and activity. There was a
distinct blood plate phenotype and plaque defect for this mu-
tant (Fig. 2; Table 3). Furthermore, ?prsA2 culture superna-
tant and purified, full-length LLO from a strain containing a
transposon in prsA2 had similar decreased levels of hemolytic
activity relative to WT levels (Table 4). Finally, Western blot
analysis revealed that while the total levels of LLO exported
into the supernatant were similar to WT levels, there were
additional lower-molecular-weight species of the toxin (Fig.
5A). The amounts of these species decreased in the comple-
mented strains and drastically increased in a strain of L. mono-
cytogenes that overproduced LLO (Fig. 5A). These additional
bands did not stain with an anti-FLAG antibody when they
were purified from a prsA2 mutant expressing LLOFLAG, indi-
cating C-terminal cleavage (Fig. 5B). These results strongly
suggested that PrsA2 contributes to the proper folding of ex-
Given the additional results that strains lacking PrsA2 have
diminished lecithinase activity and a diminished ability to
swarm, it is likely that PrsA2 contributes to the folding of
several other exported proteins. We therefore hypothesize that
the decrease in LLO activity, as well as both the plaque and in
vivo defects in prsA2 mutants, arises as a result of multiple
misfolded virulence factors. Our lab is currently investigating
Previous studies had identified a link between the expression
of prsA2 and the master virulence transcription factor prfA:
prsA2 transcript levels were upregulated during intracellular
growth (9) and upregulated in bacteria grown in cytosol-mim-
icking medium (52), and PrsA2 protein was upregulated in a
prfA* background (56). Given the computationally identified
PrfA box upstream of prsA2, it was hypothesized that this gene
was directly regulated by PrfA (52). However, our results sug-
gest that PrfA does not directly regulate prsA2 expression: a
116-bp region upstream of prsA2 lacking the identified PrfA
box (Fig. 4A) was sufficient to complement the transposon
insertion ex vivo (Table 5) and the in-frame deletion both in
the hemolytic-activity assay and in vivo (Fig. 4B). While PrfA
may indirectly control prsA2 expression, there appear to be
environmental conditions unassociated with virulence, such as
VOL. 191, 2009 ROLE OF PrsA2 IN LISTERIA HEMOLYTIC ACTIVITY3961
swarming, under which prsA2 expression is independent of
PrfA (Fig. 6B).
During infection, L. monocytogenes undergoes dynamic
changes in gene and protein expression (9). This includes the
translation of a variety of different PrfA-regulated genes within
a short period of time: the genes for internalins (InlA and
InlB) to promote uptake into a cell; LLO, the phospholipases
C (PI-PLC and PC-PLC), and Mpl to promote phagosome
escape; and then ActA to hijack host cell actin (84). The
PrfA-regulated genes comprise the most abundant secreted
proteins during this transition (71; data not shown).
Interestingly, the effect of the prsA2 mutants on secreted
proteins became more pronounced the more LLO was se-
creted (Fig. 5C, lanes 2, 4, and 8); the presence of the addi-
tional bands noted in the legend of Fig. 5 is most pronounced
in the LLOS44Abackground, followed by the WT background
and then the ?hly background. We therefore hypothesize that
L. monocytogenes PrsA2 is necessary for the bacterium during
times of increased export stress. This includes intracellular
growth when multiple virulence factors—especially LLO—are
produced and secreted in large quantities. Export of these
factors occurs through the Sec machinery. Entry and passage
through the Sec translocation channel requires substrate pro-
teins to be in a primarily unfolded state (82). Folding at the
trans side of the membrane is then facilitated by a number of
chaperones known as the foldases and include peptidyl-prolyl
cis/trans isomerases (of which PrsA2 is a member) (82). Accu-
mulation of unfolded LLO in the space between the extracy-
toplasmic side of the membrane and the cell wall in the mu-
tants lacking prsA2 may lead to either protein degradation or
the release of misfolded toxin, resulting in the decrease in
hemolysis in vitro (Fig. 2; Table 4) and the virulence defect in
vivo (Fig. 4). It is likely that other virulence factors, such as
PC-PLC (Fig. 6A), are similarly affected, although whether
these factors interact with PrsA2 directly remains unclear. Pre-
cedence for this model exists in B. subtilis, where the expres-
sion of chaperones has been shown to increase in response to
extracytoplasmic secretion stress (although those studies were
performed with a mutant prsA background) (30, 87).
Our hypothesis may explain results from other studies.
Gram-positive bacteria have a quality control system for ex-
ported factors within the extracytoplasmic space between the
cell membrane and peptidoglycan layer (82). One system in-
volves the proteolytic digestion of misfolded proteins by extra-
cytoplasmic proteases, such as the HtrA family of proteases
(11, 82). A recent study found that L. monocytogenes HtrA
levels increased in prfA* strains (56), and HtrA has been shown
to contribute to virulence (77, 88). Our hypothesis is consistent
with a model that, under increased export stress, including that
experienced upon infection, L. monocytogenes upregulates the
expression of several factors, including multiple external chap-
erones such as PrsA2 to manage this stress. These chaperones
may prove to be potential targets for therapeutic interventions.
We thank C. Sassetti and E. Rubin for providing the hyperactive C9
Himar1 transposase and the transducing phage phiMycoMarT7, N.
Meyer-Morse for her generous assistance with animal work, N. Wang
for her assistance with statistical analyses, R. Calendar for help with
transductions, and J. D. Sauer and G. Crimmins for critical reading of
the manuscript. We also thank H. Shen and J. Miller for the gift of
DP-L4317 and D. Higgins for the gift of Pspac(hy) and numerous other
vectors and gratefully acknowledge the construction of strain DP-
L4361 by A. Decatur.
This research was supported by National Institutes of Health grants
AI52154 (H.M.) and AI27655 and P01 AI063302 (D.A.P.). D.A.P. is a
Senior Scholar Awardee at the Ellison Medical Foundation. J.H.L. is
a Damon Runyon fellow supported by the Damon Runyon Cancer
Research Foundation (DRG 1801-04).
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