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: email@example.com.
† 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.
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