Molecular Microbiology (2015) 96(3), 437–447 ■
doi:10.1111/mmi.12918
First published online 30 January 2015
The phage tail tape measure protein, an inner membrane
protein and a periplasmic chaperone play connected roles in
the genome injection process of E. coli phage HK97
Nichole Cumby,1,2 Kelly Reimer,1,2
Dominique Mengin-Lecreulx,3 Alan R. Davidson,1,4*
Karen L. Maxwell2†
1
Department of Molecular Genetics, 2Donnelly Centre
for Cellular and Biomolecular Research and
4
Department of Biochemistry, University of Toronto,
Toronto, ON, Canada.
3
Laboratoire des Enveloppes Bactériennes et
Antibiotiques, IBBMC, UMR 8619 CNRS, Université
Paris Sud, Orsay Cedex, France.
Summary
Phages play critical roles in the spread of virulence
factors and control of bacterial populations through
their predation of bacteria. An essential step in the
phage lifecycle is genome entry, where the infecting
phage must productively interact with the components of the bacterial cell envelope in order to transmit its genome out of the viral particle and into the
host cell cytoplasm. In this study, we characterize this
process for the Escherichia coli phage HK97. We have
discovered that HK97 genome injection requires the
activities of the inner membrane glucose transporter
protein, PtsG, and the periplasmic chaperone, FkpA.
The requirements for PtsG and FkpA are determined
by the sequence of the phage tape measure protein
(TMP). We also identify a region of the TMP that mediates inhibition of phage genome injection by the HK97
superinfection exclusion protein, gp15. This region of
the TMP also determines the PtsG requirement, and
we show that gp15-mediated inhibition requires PtsG.
Based on these data, we present a model for the in
vivo genome injection process of phage HK97 and
postulate a mechanism by which the inhibitory action
of gp15 is reliant upon PtsG.
Accepted 22 December, 2014. For correspondence. *E-mail
[email protected]; Tel. (+1) 416 978 0332; Fax (+1) 416 978
6885. †E-mail [email protected]; Tel. (+1) 416 978 0872;
Fax (+1) 416 978 8287.
© 2014 John Wiley & Sons Ltd
Introduction
Bacteriophages (phages), the viruses that infect and kill
bacteria, are the most numerous biological entities on
Earth. They are found everywhere that bacteria reside,
including soil (Marsh and Wellington, 1994; Kimura et al.,
2008), water (Suttle, 2007) and the human gastrointestinal tract (Breitbart et al., 2003; Mills et al., 2013). Because
phages play significant roles in the control of bacterial
populations, as well as the spread of virulence factors
(Suttle, 2007) and antibiotic resistance genes (Marti et al.,
2014), a thorough understanding of mechanisms by which
these viruses spread among bacteria is critical.
The non-contractile tailed siphophages comprise the
largest group of phages (Ackermann, 2007). For a
siphophage to productively infect a new bacterial cell, it
must inject its genome through the multilayered cell envelope. The process by which this occurs is not well understood. In a Gram-negative bacterial cell, the phage
genome must pass through the outer membrane, periplasmic space, peptidoglycan layer and inner membrane
to gain entry to the bacterial cytoplasm where replication
can begin. This process begins with the recognition of a
receptor on the cell surface, such as lipopolysaccharide or
an outer membrane porin. Studies with phage λ have
revealed that the outer membrane protein LamB acts as
the phage receptor, but the bacterial inner membrane
PtsM complex also plays a role in the infection process
(Scandella and Arber, 1976). Other phages have been
shown to depend on the presence of different proteins in
the cell envelope. For example, phages C1 and C6 use
the outer membrane proteins BtuB and FhuA, respectively, in addition to the inner membrane and periplasmic
proteins DcrA and DcrB (Samsonov and Sineoky, 2002).
Like phage C6, phage T1 uses FhuA as a receptor, but it
requires the activity of TonB (Hancock and Braun, 1976),
which is anchored to the periplasmic side of the inner
membrane (Postle and Skare, 1988; Hannavy et al.,
1990; Roof et al., 1991). These data illustrate that even
within the common Escherichia coli host, phages use a
variety of different proteins to traverse the layers of the
cell envelope during genome injection.
438 N. Cumby et al. ■
The phage tail tape measure protein (TMP) has also
been implicated in genome injection. In vitro studies with
phage λ revealed that the presence of LamB in liposomes
resulted in the TMP extruding from the tail and associating
with the liposome (Roessner and Ihler, 1984). This process
allowed ions, but not proteins, to traverse the membrane
(Roessner and Ihler, 1986). These data suggested that the
TMP forms a channel though the host cell membrane that
can be used for phage genome entry. Cryo-electron tomography studies of the E. coli podophage T7 (Hu et al., 2013)
and siphophage T5 (Bohm et al., 2001) revealed channellike structures emanating from these phages through the
bacterial cell envelope, providing in vivo support for this
model. The mechanism(s) by which phage proteins form
this channel and interact with host proteins to mediate
genome entry are unclear.
Phage genome injection can be blocked by the activity
of superinfection exclusion proteins, which protect the
bacterial host population from death by invading phages
(Labrie et al., 2010). Study of the mechanism(s) by
which this class of superinfection exclusion proteins
inhibits phage infection provides an excellent opportunity
to gain knowledge of the process by which phage
genomes enter bacterial cells. The Ltp protein from
the Gram-positive Streptococcus thermophilus phage
TP-J34 is a membrane-associated lipoprotein that interferes directly with phage genome injection. A mutation
that allowed TP-J34 to overcome this superinfection
exclusion phenotype is mapped to the TMP, suggesting
that Ltp blocks its proper function (Bebeacua et al.,
2013). Recently, we discovered a superinfection exclusion protein encoded by phage HK97, called gp15, that
acts in a similar manner and blocks genome injection by
phages HK97 and HK75 (Cumby et al., 2012). We determined that gp15 activity was mediated through a step
downstream of adsorption, and likely functioned through
the tail tube or TMP (Cumby et al., 2012). Thus, it
appears that the superinfection exclusion proteins from
the Gram-positive S. thermophilus phage TP-J34 and
the Gram-negative E. coli phage HK97 may share a
common mode of action.
In this study, we further investigated the DNA injection
process of phage HK97 with the goal of gaining insight into
the mechanisms of action of gp15 and other factors
involved in this process. We utilized a high-throughput
screen to identify two new host factors required for HK97
DNA injection: the FkpA periplasmic chaperone and the
PtsG inner membrane sugar transporter. We then showed
that the requirement for these proteins is mediated through
specific regions of the HK97 TMP. We also discovered that
the superinfection exclusion activity of gp15 is linked to the
requirement for PtsG. These data have provided new
general insights into the siphophage genome injection
process.
Results
Bacteriophage HK97 requires the host proteins FkpA
and PtsG for genome entry
To identify E. coli proteins that are involved in HK97
phage genome entry and might interact with gp15, we
tested the plaquing efficiency of HK97 on a collection of
815 E. coli strains from the Keio collection (Baba et al.,
2006). These strains contained single deletions in nonessential genes known or predicted to encode proteins
involved in synthesizing components of the cell envelope,
or that are localized to the cell envelope (i.e. the inner
membrane, outer membrane and periplasmic space). We
identified three strains, ΔlamB, ΔfkpA and ΔptsG, that did
not support growth of HK97. LamB was previously identified as the outer membrane receptor of HK97 (Dhillon
et al., 1980a; Juhala et al., 2000). PtsG and fkpA encode
an inner membrane glucose transporter (Buhr et al.,
1994) and a periplasmic peptidyl-prolyl cis/trans isomerase folding chaperone (Horne and Young, 1995) respectively. Neither of these proteins was previously known to
be required for replication of HK97 or any other phage.
We examined the ability of other phages in our collection
to plaque on ΔfkpA and ΔptsG, and determined that
phage HK446 required FkpA and HK544 (Dhillon et al.,
1980b) was partially dependent on FkpA (Table 1). Phage
λ, which uses the same outer membrane receptor as
HK97 but is not dependent on FkpA or PtsG, was able to
form plaques on these mutants with wild-type titers. This
illustrated that the receptor for HK97 is present and functional on the bacterial cell surface, and the block to HK97
infection mediated by these mutants is at a step downstream of phage adsorption.
To ascertain whether deletion of ptsG and fkpA prevented the passage of the HK97 genome through the cell
envelope, we directly assayed the ability of the phage
genome to enter the host cell using a potassium efflux
assay. The infection of a susceptible bacterial strain by a
phage leads to a transient efflux of potassium ions from
inside the cell into the surrounding medium in a process
that can be monitored using a potassium-selective electrode (Boulanger and Letellier, 1992; Letellier et al.,
1999). When HK97 was mixed with BW25113 cells, the
parent strain of the Keio collection (Baba et al., 2006), we
observed robust potassium efflux (Fig. 1). As expected,
no efflux was observed when HK97 was mixed with the
ΔlamB mutant. Both the ΔfkpA and ΔptsG strains showed
markedly decreased levels of potassium efflux when
mixed with HK97, indicating that the products of these
genes are involved in the injection of the HK97 genome.
Interestingly, both ΔfkpA and ΔptsG consistently released
slightly more potassium than ΔlamB (Fig. 1). This suggested that the genome entry process may have partially
proceeded, likely breaching the outer membrane via the
© 2014 John Wiley & Sons Ltd, Molecular Microbiology, 96, 437–447
Phage and host protein requirements for HK97 genome injection 439
Table 1. Cell envelope proteins required for genome injection.
Phage titer (pfu ml−1)
Phage
HK97
HK446
HK544
HK97446-TMP
HK97544-TMP
HK54497-TMP
λ
WT
9
10
109
109
109
109
109
109
ΔlamB
0
109
109
0
0
109
0
ΔfhuA
9
10
0
0
109
109
0
109
ΔptsG
ΔfkpA
WT + gp15
0
109
109
109
109
0
109
0
0
107
0
107
0
109
0
109
109
109
109
0
109
WT, wild type cells.
interaction with LamB but stalling in the periplasm
because of the loss of the periplasmic (FkpA) or inner
membrane (PtsG) protein. This block in DNA injection is
similar to that observed when phage HK97 was mixed
with cells expressing the gp15 superinfection exclusion
protein (Cumby et al., 2012). These data suggest that the
inhibition of plaquing caused by the deletion of genes fkpA
and ptsG is downstream of phage adsorption and likely
results from a block in the passage of the DNA through the
periplasm.
The chaperone activity of FkpA is required for
HK97 infection
To elucidate the role of FkpA in phage genome entry,
we tested which of its two functions is required for HK97
infection. FkpA encodes two distinct activities; its Nterminal domain acts as a protein chaperone, while the
C-terminal domain encodes a peptidyl-prolyl cis/trans
isomerase activity (Saul et al., 2004). We plated serial
dilutions of phage HK97 on lawns of ΔfkpA cells transformed with plasmids expressing full-length FkpA, the
N-terminal domain or the C-terminal domain (Fig. 2A). As a
control, we plated serial dilutions of colicin M, which
requires both activities of FkpA for cell killing. HK97 was
able to kill cells expressing either full-length FkpA or the
N-terminal domain (Fig. 2A), demonstrating that it requires
only the chaperone activity of FkpA.
To determine if the gp15 superinfection exclusion protein
blocks HK97 infection by inactivating FkpA, we examined
the ability of the bacteriocin colicin M to kill cells expressing
gp15. The activity of full-length FkpA is needed to fold the
colicin M bacteriocin into its active conformation for cell
lysis (Hullmann et al., 2008). We found that expression of
gp15 did not affect the ability of colicin M to lyse cells,
suggesting that gp15 does not inactivate FkpA (Fig. 2B).
Further evidence that gp15 does not inactivate FkpA is
provided by the ability of an FkpA-dependent phage,
HK446, to form plaques on gp15-expressing cells
(Table 1). In summary, although the chaperone activity of
FkpA is required for HK97 infection, the inhibitory activity of
gp15 on phage HK97 plaquing is not mediated through this
protein.
FkpA and PtsG requirements for HK97 DNA injection
are mediated through the TMP
Fig. 1. Potassium efflux induced by phage HK97. Phage genome
injection into wild-type Escherichia coli by HK97 (◊) leads to efflux
of potassium out of the cell and into the surrounding medium. This
effect is abrogated in cells lacking the LamB outer membrane
receptor (+). Potassium release is also dramatically reduced when
HK97 infects ΔfkpA (▲) and ΔptsG (×) mutants, illustrating that
these factors are required for genome injection.
© 2014 John Wiley & Sons Ltd, Molecular Microbiology, 96, 437–447
Because the siphophage TMP is known to be crucial for
the DNA injection process (Scandella and Arber, 1976;
Roessner and Ihler, 1984; Katsura, 1987; Boulanger
et al., 2008), we hypothesized that variations in the HK97
TMP sequence might affect its requirement for PtsG and
sensitivity to gp15. Fortuitously, E. coli phages HK446 and
HK544 possess tails composed of proteins that share
80–90% sequence identity with HK97. The exception is
their TMPs, which contain domains that have diverged in
440 N. Cumby et al. ■
and was not inhibited by gp15. We also engineered a
mutant HK544 phage in which the TMP was replaced by
the HK97 TMP (Table 1). The introduction of the HK97
TMP into this phage made it unable to form plaques on
ΔptsG and ΔfkpA, and it became inhibited by gp15. As
expected, the TMP swaps had no effect on the outer
membrane proteins required by the mutant HK97 phages,
which are determined by the central tail fiber (Wang et al.,
1998; Berkane et al., 2006). These data show that the
TMP alone is sufficient to determine the requirements for
PtsG and FkpA, and sensitivity to gp15.
The requirement for PtsG and inhibition by gp15 are
mediated by a small region of the HK97 TMP
Fig. 2. The chaperone activity of FkpA is required for HK97
infection, but does not contribute to the superinfection exclusion
activity of gp15.
A. Bacteriophage HK97 requires only the N-terminal chaperone
domain of FkpA for infection, while colicin M requires the activity of
both domains. Serial dilutions of colicin M and phage HK97 were
plated on ΔfkpA transformed with a plasmid expressing full-length
FkpA, the FkpA C-terminal domain (CTD) or the FkpA N-terminal
domain (NTD).
B. The gp15 superinfection exclusion protein does not inactivate
FkpA. Colicin M was added to actively growing Escherichia coli
cultures and the optical density at 600 nm was monitored over a
period of 6 h using a TECAN 96-well plate reader. The scales for
each panel are the same. Wild-type E. coli (WT) was lysed by
colicin M, while the growth of ΔfkpA and ΔfhuA strains, which do
not express the two host factors required for colicin M activity, was
not inhibited. The expression of gp15 from a plasmid (pEx15) did
not inhibit colicin M lytic activity.
sequence from HK97 (Fig. 3A and B). Strikingly, we found
that phage HK446 was able to plaque with wild-type efficiency on ΔptsG cells and was not inhibited by gp15
(Table 1). However, it still required FkpA. Phage HK544
was able to form plaques on ΔptsG and gp15-expressing
cells, and plated with decreased efficiency on the ΔfkpA
strain (Table 1). To determine if the TMP alone conferred
these plating behaviors, we engineered two HK97 hybrid
phages in which the wild-type TMP was replaced by the
TMP from HK446 or HK544 and we examined their abilities to form plaques on the various mutant strains
(Table 1). When the TMP from HK446 was present in the
HK97 background, the hybrid phage was able to form
plaques on both ΔptsG and gp15-expressing cells. The
HK97 hybrid phage bearing the TMP gene from HK544
was able to form plaques on the ΔptsG and ΔfkpA strains
Because the inhibitory activity of gp15 was clearly not
mediated through FkpA, we focused further experiments
on the relationships between the roles of gp15 and PtsG in
the HK97 injection process. The TMP sequence from
phage HK97 is > 99% identical to HK446 over the N- and
C-termini, but the sequence identity falls to only 41%
between residues 452 and 745 (Fig. 3A and B). We postulated that this region might be responsible for the dependence on PtsG and sensitivity to superinfection exclusion by
gp15. To test this idea, we replaced residues 452–745 in
the HK97 TMP with the corresponding residues (residues
452–761, see Fig. 3B) from the HK446 TMP. The hybrid
phage was able to plaque on cells lacking PtsG, confirming
that this region of HK97 specifies this requirement
(Table 2). This hybrid was also able to form plaques on
cells expressing gp15; this provides definitive evidence
that the PtsG requirement and sensitivity to gp15 are
encoded in the same region of the HK97 TMP.
To more precisely define the TMP sequences that
encode gp15 sensitivity and PtsG dependence, we
created smaller TMP swaps. When residues 452–600 of
the HK97 TMP were replaced by the corresponding residues from the HK446 TMP, the hybrid phage was able to
form plaques on cells lacking PtsG and was not inhibited
by gp15. By contrast, when HK97 residues 553–627 or
residues 625–745 were replaced with the corresponding
regions from HK446, the hybrid phages exhibited the
same behavior as wild-type HK97 (Table 2). We also performed reciprocal swaps in which residues from HK97
were placed into the HK446 TMP. Swapping of HK97
residues 625–745 into HK446 produced a hybrid phage
with the same plating properties as wild-type HK446,
while the two other swaps involving HK446 (HK97 residues 452–745 and 553–627) resulted in non-viable
phages. Overall, these experiments indicate that residues
452–553 in the HK97 TMP are critical for imparting the
PtsG requirement and sensitivity to gp15 as swapping
residues 452–600 changed these properties while swapping residues 553–627 did not.
© 2014 John Wiley & Sons Ltd, Molecular Microbiology, 96, 437–447
Phage and host protein requirements for HK97 genome injection 441
Fig. 3. TMP sequences correlate with phage genome entry requirements.
A. The TMPs of phages HK544 and HK446 have sequences that share > 99% sequence identity with HK97 TMP (shaded gray) at their Nand C-termini. The central regions of these proteins diverge in sequence, encoding domains that share 37–66% sequence identity with HK97.
B. Sequence alignment of the central portion of the HK97, HK446 and HK544 TMPs. The sequenced were aligned using Jalview (Waterhouse
et al., 2009) and displayed using the ClustalX color scheme (Jeanmougin et al., 1998).
To further delineate the role of the 452–553 region of the
HK97 TMP, we created seven different deletion mutants
across this region and tested their in vivo function using the
plasmid complementation assay (Table 3). Despite the
small size of these deletions, only the mutant with residues
466–486 deleted was able to form any plaques on wildtype cells and the plaquing efficiency was reduced by
100-fold. This mutant was still dependent on PtsG and was
inhibited by gp15, which further narrowed the region specifying these properties to residues 487–553. All of the TMP
Table 2. Plating behaviors of HK97 and HK446 phages encoding hybrid tape measure proteins.
Phage titer (pfu ml−1)
Tape measure
backbone
Amino acids
deleted
Amino acids
inserted
WT
ΔptsG
ΔfkpA
WT + gp15
HK97
None
452–745
553–627
625–745
452–600
None
452–761
553–629
638–761
452–616
109
107
109
107
107
0
107
0
0
107
0
0
0
0
0
0
107
0
0
107
HK446
None
452–761
553–629
638–761
None
452–745
553–627
625–745
109
0
0
109
109
n/aa
n/aa
109
0
n/aa
n/aa
0
109
n/aa
n/aa
109
a. Not assessed as mutant did not produce infectious particles.
WT, wild-type cells.
© 2014 John Wiley & Sons Ltd, Molecular Microbiology, 96, 437–447
442 N. Cumby et al. ■
Table 3. Plating behaviors of HK97 tape measure protein deletion mutants.
Phage titer (pfu ml−1)
Deletion
466–486
487–507
489–519
499–519
508–528
516–532
529–549
Number of
amino acids
21
21
31
21
21
17
21
TEM phenotype
Complete
Complete
Complete
Complete
Complete
Complete
Complete
phages
phages
phages
phages
phages
phages
phages
WT
10
0
0
0
0
0
0
7
ΔptsG
WT + gp15
0
n/aa
n/aa
n/aa
n/aa
n/aa
n/aa
0
n/aa
n/aa
n/aa
n/aa
n/aa
n/aa
a. Not assessed as mutant did not produce infectious particles.
TEM, transmission electron microscopy; WT, wild-type cells.
Table 4. Plaque formation in complementation assays of TMP insertion mutations generated in the HK97 and HK446 TMPs.
Phage titer (pfu ml−1)
TMP backbone
Sequence inserted
Insertion
site
ΔptsG
WT + gp15
9
0
n/aa
n/aa
0
n/aa
n/aa
109
n/aa
109
107
n/aa
0
WT
HK97
None
HK446 452–616
HK446 452–616
n/a
452
600
10
0
0
HK446
None
HK97 452–600
HK97 452–600
n/a
452
616
109
0
106
a. Not assessed as mutant did not produce infectious particles.
WT, wild-type cells.
deletion mutants tested here were able to form normal
phage particles as assessed by electron microscopy
(Fig. 4), demonstrating that these deletions do not prevent
phage assembly, and thus likely prevent plaque formation
by inhibiting DNA injection. This region of the HK97 TMP is
clearly crucial for the DNA injection process.
Insertion of residues 452–600 of the HK97 TMP into the
HK446 TMP produces a phage that is defective in the
presence of PtsG
To determine if the presence of the TMP sequence that
encodes the requirement for PtsG was sufficient to confer
this property to a phage TMP that does not require PtsG,
we engineered two insertion mutants in the HK446 TMP
(Table 4). In these mutants, residues 452–600 of HK97
were inserted at the beginning (residue 452) or end
(residue 616) of the corresponding region in HK446. The
reverse insertions into the HK97 TMP were also created to
ascertain if addition of the HK446 PtsG-independent
region could allow HK97 to infect cells in the absence of
PtsG. Each of the four TMP insertion mutants was used to
complement the HK97 TMP knockout, and plaque formation was assessed. Only one of these mutants, in which
residues 452–600 of the HK97 TMP was inserted between
Fig. 4. The HK97 TMP deletion mutants assemble full phage
particles. Examination of crude lysates of the seven deletion
mutants using negatively stained transmission electron microscopy
revealed the production of wild-type (WT) numbers of full phage
particles for each mutant.
© 2014 John Wiley & Sons Ltd, Molecular Microbiology, 96, 437–447
Phage and host protein requirements for HK97 genome injection 443
Fig. 5. The insertion of HK97 TMP residues
452–600 in the HK446 TMP inhibits phage
infection in the presence of gp15. Phage
HK97 encoding a wild-type (WT) TMP, the
TMP from phage HK446 or the TMP from
phage HK446 with the insertion of HK97
residues 452–600 at amino acid position 616
(HK97446@616) were plated on WT BW25113
cells alone or expressing gp15 from a
plasmid, as well as ΔptsG alone or expressing
gp15 from a plasmid. Serial dilutions of each
of the three phages were plated on the
strains, incubated overnight at 37°C, and
plaque formation was observed.
amino acids 616 and 617 of HK446, produced viable
phages. Remarkably, replication of this phage appeared to
be inhibited by the presence of PtsG. When plated on
wild-type cells, the titer of this mutant phage was
∼ 106 pfu ml−1, but the titer increased to ∼ 109 pfu ml−1 in
ΔptsG cells. The expression of gp15 from a plasmid
decreased plaque formation of this mutant by > 104-fold in
wild-type cells (Fig. 5). However, gp15 expression did not
inhibit this phage mutant in ΔptsG cells, indicating that
PtsG is required for this inhibition (Fig. 5).
Discussion
Phage infection of a bacterial cell is a complex process
that requires the participation of both viral and host proteins. In this work, we identified two E. coli proteins, FkpA
and PtsG, which are required for genome injection of
phage HK97, and we genetically mapped the determinants for these requirements to the phage TMP. In addition, we identified a small region of the HK97 TMP that
confers sensitivity to the HK97 gp15 superinfection exclusion protein and discovered that it overlaps with the region
responsible for the PtsG requirement. Hybrid HK97
phages that encoded regions of other phage TMPs with
diverged amino acid sequences were able to bypass the
necessity for these proteins and were not inhibited by
gp15. However, these mutants may require other unidentified cell envelope proteins for infection.
Only a small number of host inner membrane proteins
have been shown to play a role in phage infection. It is
unclear, however, whether most phages actually do not
use inner membrane receptors, or whether these receptors have not yet been identified because of the lack of
systematic searches like the one described in this study.
One phage that parallels HK97 is E. coli phage λ, which
requires the mannose transporter, PtsM, for infection
(Williams et al., 1986). While the PtsM locus is composed
of three genes (manX, manY and manZ) that are essential
for the mannose transport and fermentation activities of
PtsM, the activity of manY alone is sufficient to facilitate λ
genome entry (Williams et al., 1986). Thus, the mechanism by which this protein mediates phage genome entry
© 2014 John Wiley & Sons Ltd, Molecular Microbiology, 96, 437–447
is not the same as the mechanism by which it transports
mannose. In this work, we determined that HK97 utilizes
the inner membrane glucose transporter, PtsG. Like
PtsM, the ability of PtsG to mediate phage DNA transfer is
not linked to its sugar transporter activity as the PtsG IIB
domain must be phosphorylated by Crr for glucose transport (Meins et al., 1993), and this gene was not required
for HK97 infection (HK97 was able to form plaques on a
Δcrr strain in this study). Besides sugar transporters, the
inner membrane serine transporter, DcrA, is used for
infection by phages C1 and C6 (Likhacheva et al., 1996;
Samsonov and Sineoky, 2002). The fact that a variety of
inner membrane proteins have been shown to be required
for infection by different phages suggests that they are an
important feature for the genome entry process. However,
because phages are able to employ a variety of inner
membrane proteins, they are probably not acting as channels for the genomes to pass through. These proteins may
anchor a region of the TMP to the inner membrane, ensuring that the phage genome passes productively into the
host cytoplasm.
The periplasmic components needed for phage infection are more poorly characterized than the outer and
inner membrane requirements. Prior to this study, DcrB
was the only periplasmic protein known to be required for
phage infection (Likhacheva et al., 1996; Samsonov and
Sineoky, 2002). As the function of DcrB is unknown
beyond that its activity is coupled to the inner membrane
serine transport protein DcrA, it is difficult to postulate its
role in the phage infection process. In this study, we
determined that the chaperone activity of the periplasmic
protein FkpA was required for HK97 entry. This suggests
that FkpA may play a role in restructuring the TMP or
protecting it from aggregation as it leaves the phage tail
and enters the periplasm, helping it form a channel
through which DNA can be transferred. Whether other
phages use periplasmic chaperones during the entry
process remains to be determined.
Our data demonstrate a clear link between the amino
acid sequence of the TMP and the cell envelope protein
requirements for genome entry. The exchange of residues
452–600 of HK97 with the corresponding region from the
444 N. Cumby et al. ■
HK446 TMP resulted in phages that were PtsG independent. Our deletion and swapping experiments identified a
smaller region between residues 487 and 553 that mediates the PtsG requirement. This region might be acting by
causing the TMP to operate somewhat inefficiently so that
an interaction with PtsG is necessary to facilitate TMP
function. If this were the case, deletions within this region
would result in PtsG independence. However, small
(20–30 residues) deletions within this region did not
produce PtsG independence, and all deletions but one
resulted in defective phage particles (Table 3), indicating
a crucial role for this region in DNA injection. An alternative scenario is that this region is interacting, either
directly or indirectly, with PtsG. Supporting this model, the
insertion of residues 452–600 into the HK446 (Table 4)
TMP resulted in a 103-fold plaquing defect that was
relieved when complementation was performed in a
ΔptsG background. This block to phage growth in wildtype cells is likely caused by an unproductive interaction
between the HK446 TMP and PtsG that is mediated by
the inserted HK97 residues. This unproductive interaction
is eliminated when PtsG is not present, and a wild-type
level of plaquing is observed.
Many phages have been shown to encode superinfection exclusion proteins that block superinfecting phages by
inhibiting the infection process at the cell envelope (Kliem
and Dreiseikelmann, 1989; Lu and Henning, 1994; Hofer
et al., 1995; Mahony et al., 2008). We previously determined that HK97 gp15 acts to block infection by HK97 at a
step downstream from adsorption to the bacterial cell
surface, and that this inhibition was mediated through the
tail proteins (Cumby et al., 2012). In this work, we found
that the HK97 TMP region mediating the PtsG requirement
(residues 487–553) also conferred inhibition by gp15. The
importance of this region in the superinfection exclusion
activity of gp15 is supported by our discovery that a hybrid
phage bearing the HK446 TMP with an insertion of HK97
TMP residues 452–600 was inhibited by gp15. When this
hybrid phage was plated in the presence of gp15 on the
ΔptsG mutant, no gp15-induced inhibition was observed.
This result implies that the presence of PtsG is required for
gp15 to exert its inhibitory effect. Furthermore, gp15 is
clearly not functioning by simply blocking the activity of
PtsG because the hybrid phage does not require PtsG for
infection, yet is still blocked in the presence of gp15 and
PtsG. We conclude that HK97 TMP residues 487–553
interact with PtsG, directly or indirectly, and that gp15
inhibits DNA injection by driving the TMP-PtsG complex
into a dead-end non-productive conformation.
The work presented in this study highlights the key roles
that the phage TMP and host proteins play in the phage
DNA injection process. We propose a model for HK97
genome injection in which interactions with LamB in the
outer membrane anchor the viral particle to the cell
Fig. 6. Model of the HK97 genome injection process. The HK97
phage tail tip interacts with LamB on the bacterial cell surface, then
the TMP exits from the tail and is inserted in the bacterial cell
envelope. The activity of the FkpA periplasmic chaperone assists
the TMP to transition to a channel-like structure. A specific
sequence in the HK97 TMP interacts with the inner membrane
protein, PtsG, and the TMP is anchored between it and LamB in
the outer membrane. This allows the TMP channel to span the
bacterial cell envelope and permits the passage of the phage DNA
from the viral capsid into the bacterial cytoplasm.
surface. The TMP is then released from the phage tail and
passes into the periplasm where the chaperone activity of
FkpA assists it in transitioning to a channel-like structure
that can transport the genome. The TMP interacts with the
inner membrane protein, PtsG, and becomes anchored
between LamB on the outer membrane and PtsG on the
inner membrane. This allows the TMP channel to span the
bacterial cell envelope, thereby protecting the phage
genome from degradation by periplasmic endonucleases
as it passes from the phage capsid into the bacterial
cytoplasm (Fig. 6). The interaction with an inner membrane
protein may be crucial for the formation of a conduit
through the inner membrane because the HK97 TMP and
others (e.g. λ and HK446) are not predicted to form transmembrane helices on their own. Whether this model, with
the required activities of outer membrane, periplasmic and
inner membrane proteins, is generally applicable to tailed
phages remains to be determined, as additional work is
needed to identify host factors that mediate the genome
entry of a variety of other phages. Our work clearly shows
that investigation of host factors involved in DNA injection
and inhibitors of the process can illuminate the mechanisms involved in this crucial step of phage replication.
Experimental procedures
Identification of siphophage genome entry factors
Single gene deletion strains from the Keio collection (Baba
et al., 2006) were grown overnight at 37°C in lysogeny broth
(LB)-Lennox broth. A 150 μl aliquot of each overnight culture
© 2014 John Wiley & Sons Ltd, Molecular Microbiology, 96, 437–447
Phage and host protein requirements for HK97 genome injection 445
was mixed with 3 ml of molten 0.7% top agar and poured onto
1.5% LB-Lennox agar plates. Three-microliter aliquots of
100-fold serial dilutions of phages in suspension medium
(SM; 100 mM NaCl, 10 mM MgSO4, 50 mM Tris at pH 7.5)
were spotted on the plates and the ability to form plaques was
assessed following incubation overnight at 37°C.
Potassium efflux assays
Potassium efflux assays were performed as previously
described (Cumby et al., 2012) using an Orion Ionplus potassium electrode (Thermo Scientific).
Colicin M activity assay
The colicin M protein was expressed from a plasmid,
pMLD188, (El Ghachi et al., 2006) in BL21(λDE3) cells
(New England Biolabs). One liter of culture was grown
at 37°C to an OD600 of 0.8, at which time isopropyl β-Dthiogalactopyranoside (IPTG) was added to a final concentration of 1 mM. The culture was incubated at 37°C for three
additional hours. The cells were collected by centrifugation,
then the cell pellet was resuspended in 30 ml of SM and
sonicated using 10 s pulses until the sample cleared. The
cellular debris was collected by centrifugation. This supernatant was employed as the colicin M stock for the killing assay.
To determine the killing efficiency of colicin M on E. coli,
wild-type BW25113, ΔfhuA, ΔfkpA and ΔlamB mutants (Baba
et al., 2006) and BW25113 cells expressing gp15 from a
plasmid (Cumby et al., 2012) were grown overnight in
LB-Lennox broth. The cells were subcultured in 100 μl of LB
in a 96-well plate and grown to an OD600 of 0.2 at 37°C in a
TECAN microplate reader. Twenty microliters of the colicin M
stock was added to each well and cell growth or lysis was
monitored by OD600.
FkpA complementation assays
The Keio single gene deletion of fkpA was complemented by
plasmids that expressed the FkpA N-terminal domain
(pTfkpNL) (Arie et al., 2001), C-terminal domain (pTfkpΔCt)
(Saul et al., 2004) or full-length FkpA (pTfkp) (Arie et al.,
2001). One hundred microliters of overnight culture was
added to 3 ml of 0.7% molten top agar and poured onto 1.5%
agar plates. Three microliters of 100-fold serial dilutions of
HK97 and 3 μl of threefold serial dilutions of a 0.1 mg ml−1
stock of colicin M (El Ghachi et al., 2006) were spotted on the
lawns and the plates were incubated overnight at 37°C.
Construction of hybrid HK97 phages encoding
TMP swaps
We first deleted the entire TMP gene (gene 16). To do this, we
cloned the coding region of gene 16 plus 300 base pairs (bp)
upstream and downstream (nucleotides 8135–12 004) into
plasmid pAD100 (Davidson and Sauer, 1994). We then
replaced the gene 16 coding region with a kanamycin resistance cassette and this plasmid was used to transform 594
cells harboring an HK97 lysogen. The cells were grown to an
© 2014 John Wiley & Sons Ltd, Molecular Microbiology, 96, 437–447
OD600 of 0.6 at 37°C and phage production was induced by the
addition of mitomycin C. Following lysis, the cell debris was
collected by centrifugation and the resulting lysate was used to
infect 594 cells. These cells were plated on LB agar supplemented with 50 μg ml−1 kanamycin and incubated overnight at
37°C to select for HK97 lysogens in which gene 16 had been
replaced with the kanamycin resistance cassette. The gene
replacement was confirmed by DNA sequencing.
To create the hybrid phages in which regions of the TMP
were replaced by other TMP sequences, the plasmid
encoding gene 16 plus 300 bp upstream and downstream
was linearized by polymerase chain reaction (PCR), with
the linear ends of the plasmid corresponding to the desired
insertion site. The insertion sequence plus 15 bp complementary to the ends of the HK97 TMP sequence was amplified by PCR and inserted into the plasmid backbone using
In-fusion HD technology (Clontech). Plasmid sequences
were confirmed by DNA sequencing. The plasmids were
transformed into the TMP kanamycin replacement lysogen,
the lysogen was induced by the addition of mitomycin C
and the resulting lysates were plated on E. coli BW25113.
Plaques were isolated and propagated, and the mutations
were confirmed by DNA sequencing. The dependence of
the hybrid HK97 TMP mutants on specific E. coli entry
factors was determined as described above.
Construction of TMP insertions and deletions
To create the plasmids used in the complementation assays
for the HK97 TMP insertion and deletion mutants, DNA encoding genes 16 and 17 (nucleotides 8435–12 045) was cloned
into pAD100 (Davidson and Sauer, 1994) under the control of
the pTrc promoter. To create the TMP insertions, this plasmid
was linearized by PCR with the ends of the plasmid corresponding to the desired insertion site. The sequence to be
inserted plus 15 bp complementary to the open ends of the
linearized plasmid was also amplified by PCR. These two
pieces of DNA were combined using In-fusion HD technology.
To create the deletion mutants, the plasmid was amplified by
PCR minus the region to be deleted. Fifteen bp complementary sequences were designed at each end of the linearized
plasmid flanking the desired deletion site. The plasmid template was re-circularized using In-fusion HD technology (Clontech). The correct placement of each insertion and deletion
was confirmed by sequencing.
Functional assay of TMP insertions and deletions by
plasmid complementation
To assay the activity of each mutant TMP, cells containing a
lysogen of the HK97 TMP knockout were transformed with a
plasmid expressing the mutant TMP. The cells were grown to
an OD600 of 0.6, induced with 1 mM mitomycin C and serial
dilutions of the resulting lysates were plated on lawns of
E. coli BW25113, ΔfhuA, ΔfkpA, ΔlamB, (Baba et al., 2006)
and BW25113 cells expressing gp15 from a plasmid. Each of
these strains was also expressing the corresponding TMP
mutant from a plasmid to allow complementation. The titer of
each phage mutant was assessed following incubation overnight at 37°C.
446 N. Cumby et al. ■
Gp15 superinfection exclusion assays
To enable expression of gp15 and mutant TMPs in the same
cell, gene 15 (GeneID: 1262549) of bacteriophage HK97 plus
57 bp upstream from its start codon and 43 bp downstream
from its stop codon was cloned into pCDFDuet-1 (Novagen).
The plasmid was sequenced and expression of gp15 was
confirmed by plating serial dilutions of HK97 on E. coli
BW25113 cells transformed with this plasmid.
Transmission electron microscopy
HK97 lysates were filter sterilized through a 0.2 μm membrane and applied to carbon coated G1400 copper grids
(Canemco, Quebec, Canada). For each lysate, 5 μl was
applied to the grid. After rinsing three times in water, the grids
were stained with a 2% uranyl acetate solution. The samples
were visualized using a Hitachi H-7000 transmission electron
microscope (Hitachi, Tokyo, Japan) at an acceleration voltage
of 75.0 kV. Data were acquired using an AMT 1K digital
camera (Advanced Microscopy Techniques, Corp., Massachusetts, USA). All images were taken at a magnification of
80 000 times.
Acknowledgements
The authors would like to thank Rodney King (Western Kentucky University) for his gift of phages HK446 and HK544, as
well as Dominik Refardt (Zurich University of Applied Sciences) and Andrew Kropinski (University of Guelph) for providing us with the phage sequences. We also wish to thank
Jean-Michel Betton for providing the FkpA-expressing plasmids used in this study. We would like to thank Diane Bona for
technical assistance and Paul Sadowski for critical reading of
the manuscript. This work was supported by Operating Grants
from the Canadian Institutes for Health Research to K.L.M.
(Fund No. MOP-62796) and A.R.D. (Fund No. MOP-115039)
and from the Agence Nationale de la Recherche to D.M.-L.
(Fund No. ANR-07-MIME-020). N.C. was supported by a
Natural Sciences and Engineering Research Council of
Canada (NSERC) CGS-D scholarship.
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