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Phage single-gene lysis: Finding the weak spot in the bacterial cell wall
Karthik Chamakura1,2 and Ry Young1,2*
From the 1Department of Biochemistry and Biophysics, Texas A&M University, College Station TX
77843-2128; 2Center for Phage Technology, Texas A&M AgriLife Research, Texas A&M University
College Station TX 77843-2128
Running title: Phage Sgl proteins target peptidoglycan biosynthesis
*To whom correspondence should be addressed: Ry Young: Department of Biochemistry and
Biophysics, Texas A&M University, College Station TX 77843-2128; ryland@tamu.edu; Tel.(979)845-
2087.
Keywords: Single-gene lysis, phage, peptidoglycan biosynthesis, MurA, MraY, MurJ, flippase,
autolysin, chaperone, antibiotic
_____________________________________________________________________________________
ABSTRACT
In general, the last step in the vegetative cycle of
bacterial viruses, or bacteriophages, is lysis of the
host. Double-stranded DNA phages require
multiple lysis proteins, including at least one
enzyme that degrades the cell wall (peptidoglycan).
In contrast, the lytic ssDNA and ssRNA phages
have a single lysis protein that achieves cell lysis
without enzymatically degrading the peptidoglycan
(PG). Here, we review four “Single-gene lysis” or
Sgl proteins. Three of the Sgls block bacterial cell
wall synthesis by binding to and inhibiting several
enzymes in the PG precursor pathway. The target
of the fourth Sgl, L from bacteriophage MS2, is still
unknown, but we review evidence indicating that it
is likely a protein involved in maintaining cell wall
integrity. Even though only a few phage genomes
are available to date, the ssRNA Leviviridae are a
rich source of novel Sgls, which may facilitate
further unraveling of bacterial cell wall
biosynthesis and discovering new antibacterial
agents.
________________________________________
INTRODUCTION TO SMALL LYTIC
PHAGES AND “SINGLE-GENE LYSIS”
By definition, the lytic bacteriophages
encode proteins for disruption of the host envelope.
The large dsDNA phages, the Caudovirales, have
multiple lysis proteins, including holins,
endolysins, and spanins, targeting the cytoplasmic
or inner membrane (IM), peptidoglycan (PG) and
outer membrane (OM), respectively, as well as
multiple proteins that regulate the lysis process
(1,2). In contrast, the small lytic phages of Gram-
negative hosts, comprising the ssDNA
(Microviridae) and ssRNA (Leviviridae), achieve
host lysis by a single gene, encoding a protein
lacking any PG degrading activity (3). This review
exclusively focuses on these Single-Gene Lysis
(Sgl) proteins of small lytic phages.
Bacterial cell wall structure and biosynthesis
An exploration of Sgl mechanisms requires
a brief review of the structure of the Gram-negative
cell wall and its biosynthesis. The key to the
structure and shape of the cell envelope is the PG
layer, consisting of 2-3 layers of glycan strands
made up of repeating disaccharide units of
MurNAc-pentapeptide and GlcNAc, cross-linked
by peptide bridges between pentapeptide side
chains of MurNAcs of adjacent strands (Fig. 1a) (4-
6). The PG has considerable tensile strength (3-300
MPa), allowing the cell to tolerate high internal
osmotic pressures (3-10 atm) while maintaining
shape (7, 8). The entire PG of a cell can be isolated
as a single complex polymer, the sacculus, studies
of which have revealed that the glycan chains run
almost perpendicular to the long axis of the cell (9).
In most Gram-negative bacteria, the PG layer is
covalently linked through >105 peptide linkages to
the C-terminal Lys residue of the major lipoprotein,
Lpp, the PG-linked Lpp is anchored almost
exclusively in the inner leaflet of OM (5, 10, 11).
Biosynthesis of the PG can be divided into
cytoplasmic, membrane, and periplasmic stages.
There are 7 enzymatic steps in the cytoplasm,
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beginning with the transfer of an enolpyruvyl
moiety from PEP to UDP-GlcNAc, catalyzed by
MurA (Supp. Fig. 1) (12). After reduction of the
enolpyruvyl moiety by MurB to create UDP-
MurNAc, the next five enzymes are involved in
adding amino acids that form the pentapeptide [L-
Ala D-Glu m-Dap D-Ala D-Ala] to the lactyl group,
resulting in the final soluble intermediate UDP-
MurNAc-pep5 (Fig. 1a) (5, 13). The first
membrane-linked step in PG synthesis begins with
the transfer of this sugar nucleotide pentapeptide to
the lipid carrier undecaprenyl phosphate (C55-P or
UndP). This reaction is catalyzed on the
cytoplasmic face of the IM by the integral
membrane protein MraY to generate a
monosaccharide-lipid compound, Lipid I (Fig. 1a
and Supp. Fig. 2a). MurG then catalyzes the
addition of a second sugar moiety (UDP-GlcNAc),
resulting in the final precursor, Lipid II (Fig. 1a).
The last step of the membrane phase is the flipping
of Lipid II so that its disaccharide pentapeptide
moiety is on the periplasmic face of the membrane.
The enzyme “flippase” that effects this transfer has
been controversial until very recently. Although
FtsW was shown to flip lipid II in vitro (14,15),
several lines of evidence now support MurJ as
being the essential lipid II flippase (16-21). The
extracellular steps of PG biosynthesis utilize the
energy stored in the phosphodiester-muramic acid
bond of the flipped Lipid II and in the D-Ala-D-Ala
peptide bond to drive the glycosyl-transferase (GT)
and cross-linking reactions respectively (13). These
steps are carried out by mono- or bi- functional
penicillin binding proteins (PBPs) and recently,
RodA, a member of the SEDS superfamily, was
shown to catalyze GT reactions (22-24).
THE FIRST SGL: PROTEIN E FROM
MICROVIRUS PHIX174
Famous phage, famous gene
X174 is the founding member and genetic
paradigm of the Microviridae, which are nearly as
widespread as the Caudovirales (25). It was the
first gDNA to be completely sequenced and also to
be synthesized in vitro (26, 27). The 10 genes
include three that are embedded out-of-frame in
essential cistrons (Fig. 1b). One of these embedded
genes is E, encoding the Sgl protein in the +1
reading frame of the essential scaffolding gene D
(Fig 1b). E is famous not only for being the first
embedded gene to be discovered but also the first
gene to be subjected to site-directed mutagenesis
(26, 28). More important here is the fact that it is
the only DNA-virus Sgl and, it was the first Sgl
gene for which the lytic mechanism was firmly
established. The methods for working out its
functional pathway have been replicated for all the
other Sgl systems and thus will be reviewed here in
some detail.
Genetics clarifies E function
The E gene was cloned into a medium copy
expression plasmid and shown to support lysis after
induction (29, 30). Despite this early focus and the
availability of the cloned E gene, the lysis
mechanism of E remained controversial for two
decades (3). Early transmission electron
microscopy studies showed that cells infected with
X174 lysed as a result of septal blebs in dividing
cells, generating a morphology that was remarkably
similar to penicillin-mediated lysis (31, 32).
Reproduction of this morphology after induction of
the cloned E led to the general model that E
interfered with PG biosynthesis (30, 33). However,
based on physiological and scanning-EM studies,
other groups proposed that E functioned either by
the activation of unspecified autolytic functions
(34-36) or by the formation of polymeric
“transmembrane tunnels” that opened the
cytoplasm directly to the medium (37). This
profusion of models painted a confusing picture for
the mechanism of E lysis, primarily because all
lacked genetic evidence.
Mutational and deletion analysis of E
revealed that the lytic function requires only the
first 34 residues; lytic function was retained without
the C-terminal 57 residues, a highly basic, Pro-rich
segment, as long it was replaced by a stable
cytoplasmic domain, even -galactosidase (38, 39).
The first genetic approach to Sgl function, to be
repeated successfully for several other Sgl systems,
was done by selecting spontaneous host mutants
resistant to plasmid-borne expression of E (38).
The first E-resistance mutations turned out to be in
a single locus, designated as slyD (“sensitivity to
lysis”), which encodes a FKBP-type PPIase
(peptidylprolyl-cis-trans-isomerase) (40). Indeed,
X174 infections of slyD knockout mutants
proceed normally in every respect except lysis
never occurs and virions hyper-accumulate.
Purified SlyD was shown to accelerate folding of
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proteins limited by cis-trans peptidylprolyl
isomerization steps but its function in vivo is not
known (41). However, E was found to be highly
unstable in a slyD background, suggesting that
SlyD has a chaperone role, probably in folding the
C-terminal domain (CTD), which is rich in Pro
residues (42).
To continue pursuit of the E target, bypass
suppressor mutations were isolated as rare plaque-
formers on a slyD lawn (42). These Epos (plates on
slyD) were found to be missense alleles at the N-
terminus of E, each of which resulted in a ~10X
increase in the biosynthesis rate, thereby
compensating for the proteolytic instability of E.
This proved to be a key technological advance.
Spontaneous “Eps” host mutants (E-pos sensitivity)
that were resistant to induction of the plasmid-
borne Epos allele were selected and cross-streaked
for sensitivity to the phage. Of ~2000 survivors, all
but three retained X174-sensitivity and
presumably were defective in E expression or
plasmid copy number. Subsequent genetic
mapping and sequencing revealed that the
mutations mapped to residues (P172 and F288) in
TMDs 5 and 9 of MraY (Fig. 3). Labelling
experiments with [3H]mDAP showed that E
blocked cell wall synthesis ~20 min before lysis,
and TLC chromatography revealed depletion of
lipid-linked label and the accumulation of UDP-
GlcNAc, confirming the inhibition of MraY (43).
Based on the mutational data and membrane
localization of both proteins, Bernhardt and
colleagues proposed that E interacted with MraY
through TMD-TMD interactions that were
disrupted in the mutant alleles (Supp. Fig. 2b) (43,
44).
in vitro analysis of E-mediated inhibition of
MraY
Initial attempts to over-express full-length
E failed due to its inherent lethality, but by doing
inductions in the presence of the heterologous
MraY from B. subtilis, a His-tagged full-length E
protein was purified with a yield of 27 g of EHis6
per liter of culture (45). Using this as an
immunoblot standard, these workers determined
that E was produced at ~500 molecules/cell at the
time of lysis, in agreement with estimates from
radiolabeling and E-LacZ enzyme assays (38, 46).
An assay based on UDP-MurNAc-pentapeptide
DNS, a fluorescent analogue of UDP-MurNAc-
pentapeptide, and phytol-P, a 20 carbon analogue
of C55-P, was developed to determine kinetic
parameters in the presence and absence of E. For
both substrates, the addition of purified E had no
effect on the apparent Km but reduced the Vmax,
indicating that E is a noncompetitive inhibitor of
MraY (45). This was further supported by the
observation that overexpression of either wild-type
or the catalytically inactive allele of mraYD267N
protects cells from E-mediated lysis, presumably by
titrating out E (43, 47).
The E-MraY interaction
Some details of the interaction between E
and MraY have been determined by assessing lysis
kinetics in bulk culture in the context of mutant E
alleles, both from selections and site-directed
mutagenesis, and both wt and catalytically inactive
MraY (43-45, 47). Despite near saturating
mutagenesis, mutations in only five mraY positions
yielded E-insensitive phenotypes: one in TMD5
(G186S), two in TMD9 (F288L and V291M) and
two (P170L; L172) the periplasmic loop above
TMD5 (47). By testing these alleles in context of a
catalytically inactive MraYD267N for the ability to
protect against E lysis in an mraY+/mraYD267N
merodiploid, the alleles were grouped into 3 classes
of apparent affinity for E in vivo, with G186S and
V291M ranking with the wt at the highest affinity,
F288L and the B. subtilis version of MraY the
lowest, and the P170L and P172 alleles
intermediate (47). This classification was reflected
in the in vitro inhibition assays (45). Based on the
crystal structure of MraY from Aquifex aeolicus,
the TMD5 and TMD9 sites are clustered on the
same outside face of the molecule within the
periplasmic leaflet of the IM (Fig. 2 ) (48). TMD9
is split into two helical fragments: the N-terminal
9a, within which both E-insensitivity mutations
map, and the C-terminal 9b, which does not pack in
the helical bundle but is splayed out nearly laterally
in the bilayer. The loop mutations map to a beta
hairpin structure, with one directly above TMD5
(L172) and the other just above TMD9 (P170).
Assuming these mutations reflect critical contacts
in the E binding site, the spatial arrangement
indicates that the N-terminal segment of the TMD
of E makes those contacts. This notion is consistent
with the results from the Clemons group, who
constructed a large set of alanine and leucine
substitutions as well as truncation alleles, focused
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on the TMD of E (49). This collection was analyzed
by induction in vivo, monitoring the kinetics of
lysis and the accumulation of the E protein in the
membrane fraction. Mutations that affected lysis
without affecting accumulation mapped to a single
face of the TMD, suggesting that this face interacts
with the two TMDs of MraY that housed the E-
resistant changes. Also, the TMD as a whole
appears to be relatively insensitive to changes in its
C-terminal segment, which can be replaced by a
homopolymer of Leu residues. Moreover, Phe
substitutions at Leu19 and Leu23, both of which are
on the same helix face as the inactivating mutations
and one of which (L19F) corresponds to an original
Epos mutation, enhance lytic function without
enhancing membrane insertion. Using IMAC-
chromatography and Western blotting, complexes
of E and MraY could be demonstrated. However,
the efficiency of MraY recovery did not correlate
well with lytic function of the E allele used,
indicating that simple binding of E to MraY is not
sufficient for inhibition. Overall, the details of how
E binds MraY and interferes with Lipid I synthesis
will likely require a structure of the E-MraY
complex.
OVERVIEW OF SGL GENES IN SSRNA
PHAGES
The Sgl proteins undoubtedly evolved
because of the extremely constrained size of the
phage genome; compared to the Microviridae, this
constraint is even worse for the ssRNA phages (the
Leviviridae, or the leviviruses), which have ~4 kb
gRNAs and only three core genes (Fig. 1b).
Lacking tail structures, leviviruses exploit
retractable pili to initiate infection. By far the best
studied leviviruses are MS2 and Q, both specific
for the canonical F conjugation pilus; many other
F-specific leviviruses that are related to these two
paradigms have been studied (50-55). However,
seven other distinct leviviruses are known, each
targeting a different retractable pilus (Supp. Table
1) (56-61). The Sgl genes have been identified in
eight of the nine distinct leviviruses by showing
that, like for E, induction of a plasmid-borne clone
is necessary and sufficient for lysis. Importantly,
all cause disruption of the cell wall by accessing
different cellular targets, suggesting that in each
case, a new Sgl was evolved after radiation to a new
retractable pilus, no doubt facilitated by the
extremely high mutation rate of the RNA-
dependent replicase (62). This means that even
with the low total genomic database of less than
50,000 bases of unique Leviviridae genomes, there
are multiple Sgl systems which might be exploited
for probing the biosynthesis and dynamic
homeostasis of the cell wall. In the following, the
Sgl systems of Q, M and MS2 will be reviewed.
The order is not chronological but makes sense in
that the targets of the first two Sgl proteins have
been identified, whereas the MS2 Sgl system is an
enduring mystery.
THE “PROTEIN ANTIBIOTIC”: A2
FROM QBETA
Finding rats
The A2 protein has multiple functions
during Q infection: it functions in virion assembly
(63), is bound to and provides protection for the
gRNA against RNase degradation (64), mediates
interaction with the F-pilus, and is internalized into
the host cytoplasm along with the genomic RNA
(63-65). Remarkably, A2 also functions as the Sgl
protein. The lytic activity of A2 was first
demonstrated by Winter and Gold in 1983, who
showed that induction of A2 cloned on a medium-
copy plasmid is necessary and sufficient to cause
lysis (66).
To identify the target of A2, the same method was
used as for X174 E, selecting for host mutants that
survived induction of a plasmid-borne A2 gene,
followed by screening survivors by cross-streaking
with Q phage and the mutants that passed
selection/screen were designated as rat (resistant to
A-two) mutants (67). Genetics and sequence
analyses revealed a single missense change in
murA, L138Q. As with E, the incorporation of [3H]
mDAP label into PG is blocked at least 20 min
before the onset of lysis in cells induced for A2.
Biochemical analysis of the sugar nucleotide pool
from A2-inhibited cells revealed that UDP-GlcNAc
was elevated, confirming MurA as the target. In
vitro inhibition of MurA by purified A2 could not
be demonstrated, mainly because overexpressed A2
was insoluble. However, in what seems to be the
only instance of using virions for enzyme
inhibition, it was shown that the catalytic activity of
MurA, but not MurAL138Q, in crude extracts could
be blocked by the addition of highly purified Q
virions. Later experiments done with purified
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MurA and Q particles confirmed these results
(68). Based on the turnover number of MurA and
the number of purified virions needed to block its
enzymatic activity, the MurA-A2 dissociation
constant was determined to be ~10 nM (67).
The A2-MurA interaction
In addition to in vitro inhibition assays,
direct protein-protein interaction between A2-MurA
and A2 -MurAL138Q was also demonstrated by yeast-
two-hybrid analysis, with the latter pair displaying
a weaker signal, suggesting that the mutant allele
weakens A2 binding (68). To probe the interaction
of MurA with A2 in vitro, the well-characterized
catalytic pathway of MurA was exploited. The
MurA reaction is well ordered, with UDP-GlcNAc
binding in the catalytic cleft associated with a
dramatic shift from open to closed conformation,
which then allows PEP binding and catalysis.
Interaction studies were done using a soluble MBP-
A2 fusion protein and various forms of MurA,
including the original rat allele, MurAL138Q, and
MurAD305A, which is disabled for catalysis but not
substrate binding, and with various combinations of
the substrates and the suicide inhibitor fosfomycin.
The results clearly showed that A2 preferentially
binds to the UDP-GlcNAc-liganded, closed form
MurA, preventing PEP from binding. Details of the
binding surface were obtained by site-directed
substitutions of amino acids in the area around
L138, yielding a cluster of new rat alleles that
blocked Q plaque formation and clearly defined
an interaction surface surrounding the catalytic
loop, including the catalytic domain, CTD and the
catalytic loop (Supp. Fig. 3).
A2 differs significantly from the Mat
proteins of MS2 and related Leviviridae, especially
in the N-terminus; deletion analysis confirmed that
the lytic function is fully defined in the first 180
residues. To map the interaction domain, A2por
(plates on rat) suppressor alleles were isolated and
mapped to three positions (L28, D52 and E125) in
the N-terminal domain (69). However, none of the
por alleles were lytic when cloned and induced in
the rat1 host. Immunoblot analysis revealed that
A2por mutant levels increased much more rapidly
than the parental A2 during infection, resulting in
early lysis and reduced yield of progeny virions in
the wt host. Inspection of the sequence around the
por sites confirmed that the mutations disrupt
significant RNA structures that repress translational
initiation in the viral RNA, thus bypassing the
reduced A2-MurAL138Q affinity by increasing the
quantity of the phage protein.
The interaction interface was recently
resolved in asymmetric cryo-EM structures of Q
particles in complex with UDP-NAG-liganded
MurA or fosfomycin-liganded MurA (63) (Fig.
3ab). The cryo-EM structures validated the
interaction interface on MurA inferred from the
various rat alleles and also confirmed that the NTD
of A2 is in contact with MurA (Fig. 3c, d, and e).
LYSM: NEW TARGET AND SETTLING A
DEBATE
The lysis gene (lysM) of phage M has
evolved completely embedded in the +1 reading
frame of the rep gene and it encodes a 37 amino
acid protein with a single TMD (58). The functional
LysM-eGFP fusion suggests an N-out and C-in
membrane topology (70). Early insights into the
molecular mechanism of LysM lethality came from
the observations that lysis proceeded through septal
catastrophes, like A2 and E, suggesting that LysM
might be an inhibitor of cell wall biosynthesis (31,
67, 70). The identity of MurJ as the molecular target
of LysM was revealed in multi-copy suppression
experiments, where plasmids carrying murJ in
random fragments of E. coli genome suppressed
lysM lethality. Furthermore, isolation of 9
spontaneous lysM- resistant mutants that mapped to
two of the fourteen TMDs of MurJ suggested a
possible interaction interface and a plausible
mechanism (Fig. 4). Additionally, it was shown that
LysM was specific to MurJ and the cells can be
rescued from LysM lethality by the expression of
heterologous lipid II flippase Amj from B. subtilis.
To address the conformational state in
which LysM binds to MurJ, a substituted-cysteine
accessibility method (SCAM) was used (70, 71). In
the presence of LysM, SCAM analysis showed that
5 TMD positions showed altered SCAM patterns,
which suggested that LysM binding locks MurJ into
one of the two conformations proposed to constitute
the lipid II-flipping cycle. The SCAM labeling
pattern is consistent with MurJ being locked in a
“periplasmic open” conformation, which would
lead to an accumulation of lipid-linked PG
precursors in the inner leaflet of IM and a
corresponding decrease on the periplasmic side.
Both predictions were confirmed by in vivo
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flippase assays, strongly indicating that LysM
blocks MurJ’s activity (70). Moreover, in the
presence of the LysM-resistant murJ alleles, the
precursor levels were restored to normal. The fact
that LysM only targets MurJ and causes lysis
strongly suggests that MurJ is the only active lipid
II flippase in E. coli. However, the interpretation of
the LysM-resistant murJ alleles and the interaction
interface would greatly benefit from the structure of
the MurJ-LysM complex.
`
MS2 LYSIS: TO L AND BACK
L: the first autolysin?
The L gene was not recognized as a gene in
MS2 until the isolation of a plaque-forming
defective nonsense mutant that belonged to a
complementation group distinct from mat, coat and
rep (72). Subsequent radioactive labeling
experiments established L as the fourth gene of
MS2, encoding a 75 aa polypeptide (73). As had
been done with E and A2, a plasmid clone of L was
shown to cause lysis after induction and the L
protein was shown to be associated with the
membrane fraction (73, 74). Opposite to E, it is the
39 residue CTD of L that accounts for membrane-
localization and lytic function, with the N-terminal
36 highly basic residues shown to be dispensable
for lysis (Supp. Fig. 4) (75). A key experiment was
that, after induction, net murein synthesis, as
assessed by [3H] mDAP into SDS-insoluble
material, was unaffected prior to the onset of lysis
(76). This clearly differentiates L action from the E,
A2 and LysM Sgl proteins, all of which cause
cessation of cell wall synthesis by interrupting the
supply of Lipid II to the PG machinery (43, 67, 70).
To these workers, the most significant finding was
that induced L lysis was severely compromised in
acidic (pH 5.5) conditions, despite normal
accumulation of L, raising a compelling analogy to
penicillin-induced autolysis, which is also blocked
under these conditions (77). This led to a general
model in which L effected lysis by inducing
autolysis, although the precise definition thereof
was not provided. In immuno-EM experiments, L
was shown to preferentially localize to apparent
zones of adhesion between the IM and OM (78).
This association with adhesion zones was
emphasized by the fact that L lysis is also
compromised in cells that lacked the periplasmic
osmoprotectant membrane-derived-
oligosaccharide (MDO) (79). These cells were
shown to have many fewer adhesion zones and a
much wider periplasm, and in this case L appeared
to be subject to degradation. Furthermore, a
synthetic polypeptide corresponding to the C-
terminal 25 aa of L was shown to permeabilize both
liposomes and inverted membrane vesicles, leading
the authors to invoke induction of autolysis after
membrane permeabilization (80). However, these
experiments lacked a negative polypeptide control
and the experiments were done at peptide to vesicle
ratio in excess of 1000; moreover, permeabilization
and depolarization does not result in rapid autolysis
in E. coli, so the physiological relevance of these
experiments is questionable.
Back to L: genetic and molecular analysis
The consignment of MS2 L to the role of
phage-encoded autolysin seemed to end further
interest in its function, despite the likelihood that a
critical component of cell wall homeostasis was
targeted. However, over the next decades, a few
new Leviviridae were characterized, many of which
shared the same genetic architecture as MS2,
despite no significant nucleotide sequence
similarity (60, 61) (Supp. Table 1). This included
not only new leviviruses specific for the F pilus but
also against the conjugational pilus of several R-
factor plasmids, and the polar pilus of Pseudomonas
(Supp. Fig. 5) (57, 58, 60, 61). We noticed that the
L proteins, although unrelated in terms of sequence,
shared an apparent domain organization with L:
domain 1, N-terminal, highly charged; domain 2,
very hydrophobic and lacking charged residues;
domain 3, a central Leu-Ser dipeptide; and domain
4, a variable CTD (81). The conserved architecture
suggested that L-like Sgl systems widespread
among Gram-negative bacteria were all targeting
the same host function.
Two genetics-based approaches were
mounted, the first aimed at identifying host factors,
using, as before, inducible plasmid-based clones of
L (81, 82). To avoid mutations that reduced copy
number or L transcription, a blue/white reporter
plasmid was constructed and from hundreds of
colonies surviving L induction from this construct,
two blue colonies were identified and designated as
ill (insensitive to L lysis) mutants (82).
Surprisingly, the ill mutations mapped to dnaJ,
which encodes a widely-conserved chaperone
involved in the heat shock response (83). Analysis
revealed that, in both, a P330Q missense change in
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dnaJ accounted for the Ill phenotype and abolished
MS2 plaque-formation, with both phage and
survival phenotypes recessive. The Pro330 residue
is the most conserved residue in the CTD of DnaJ,
which is clearly a conserved segment although its
function is unclear. The P330Q change was found
to preserve the heat shock function of DnaJ but
abrogates the ability of DnaJ to form complexes
with L. The L suppressors, designated as Lodj
(overcomes dnaJ) alleles, were isolated as mutants
that allowed lysis in dnaJP330Q background; these
proved to be deletions of the dispensable NTD of L.
Isogenic inductions of the parental and Lodj alleles
revealed that lysis was much earlier with the
truncations. These results led to a model in which
the NTD of L has a regulatory, lysis-delay function
that blocks the interaction of L with its target; in this
model, DnaJ is required for relief of this steric block
(Supp. Fig. 6).
To identify key functional elements of L
itself, a nearly-saturated mutational analysis of L
generated a collection of 103 alleles with single
codon changes conferring absolute lysis defects
(Supp. Fig. 4) (81). The mutational distribution
validated the proposed four domain structure of the
L Sgl proteins. Domain 1, comprising the
dispensable, highly basic region, gave rise to only
one non-lytic allele (Q33H). Domain 3 containing
the LS dipeptide motif and the adjacent segments of
Domains 2 and 4 had the most missense changes
conferring non-lytic character. All of the missense
alleles tested were genetically recessive and
generated membrane-associated products of
parental size. In addition, several of the
inactivating missense changes (i.e., L44V, F47L,
F47Y, S49T, F51L and L56F) were conservative,
suggesting the L protein makes specific heterotypic
protein-protein contacts in the membrane.
Taken together, the isolation of dnaJP330Q
and mutational analysis of L both suggest that L
targets a host membrane protein, the interaction is
through the mutationally sensitive residues in
domains 2, 3, and 4, and that, like SlyD and E, a
host chaperone is involved in regulating L function.
Obviously, further investigation into the host
factors involved in L lysis are needed to understand
the mechanistic details of L function.
WHAT’S NEXT?
The premise of this review was that the
study of the Sgl systems of small lytic phages
would be interesting and lead to a better
understanding of the bacterial cell wall biosynthesis
and homeostasis. To summarize what has been
discussed, four Sgl systems have been studied in
depth, one from the microvirus X174 and three
from the Leviviridae. In three cases, the Sgl
proteins turned out to be “protein antibiotics”,
specific inhibitors of different enzymes of the
highly conserved PG biosynthesis pathway (3, 70,
84). Consideration of the available genetic and
biochemical data has already increased our
understanding of how these important enzymes
function, and in the case of LysM, settled the
identification of the flippase that exports Lipid II to
the periplasm. The next level of understanding will
come from detailed structural information about the
Sgl-enzyme inhibition complexes. The fourth case,
the L protein of MS2, has not yet been fully
characterized but appears to be the prototype of a
Sgl type that has evolved multiple times in
Leviviridae infecting a wide range of Gram-
negative bacteria (81). Genetic and biochemical
evidence was cited showing that L does not inhibit
any of the steps that lead to externalized lipid II and
its incorporation into existing PG and suggests that
it targets a host protein. There is no conceivable
answer to the L target mystery that would not be
important, possibly identifying conserved proteins
that are essential for proper coordination or
localization of cell wall synthesis machineries or
are involved in the control of powerful autolytic
enzymes.
Even with the L story still incomplete, this
seems like a pretty good haul of information from
the study of four small genes, starting with very
“low-tech”, old-fashioned and simple genetic
selections. The shocking thing is that this wealth of
molecular information is derived from the study of
only nine distinct Leviviridae (Supp. Table 1),
comprising a total of <50 kb of total genomic
information. Despite the low number, these 9
phages segregate into five different phage-type
based on where the Sgl evolved. Listed from 5’ to
3’ of the gRNA, they are: AP205 (5’ of mat); Q
(mat = A2), MS2 (overlapping end of coat and
beginning of rep), phiCb5 (middle of rep), and M
(near 3’ end of rep), (Fig. 2b). The diverse location
of the Sgl genes suggests that they have evolved
more than once and probably as a late addition to
the genome after speciation to different pili or hosts
(58, 61). (This includes all the L-like genes; none
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8
of the 6 L-type Sgls has any detectable sequence
identity, other than the LS dipeptide
sequence.) Given the diversity of Sgl systems and
the existence of multiple protein targets in PG
biosynthesis and maintenance, it is not difficult to
imagine the existence of Sgl inhibitors for every
known step in PG biosynthesis and, if L is any
indicator, possibly uncharacterized components
critical for dynamic cell wall homeostasis.
Taken together, it seems obvious that it
would be useful to identify new Sgl genes, and the
old-fashioned “phage hunt” is a reliable
approach. RNA phage hunts have so far been done
for five conjugational pili, resulting in two protein
antibiotic Sgls (A2 and LysM) and four unrelated L-
type Sgls (LMS2, LHGAL, LC1, and LPRR1). Only three non-
conjugational pili (Caulobacter, Acinetobacter and
Pseudomonas) have been targeted, resulting in two
L-type Sgls and one, Lys of Caulobacter phage
phiCB5, which does not have an L-type domain
structure but does have a single N-terminal TMD,
resembling both E and LysM. Considering the
existence of many more retractable pili systems,
there is a clear rationale to conduct RNA phage
hunts in many other systems with retractable pili,
especially in pathogenic bacteria.
Metagenomics is also having an impact. A
recent survey of publicly available RNA-inclusive
metagenomes and RNA virome studies of
invertebrate species led to the identification of ~200
new ssRNA phage genomes (85, 86). Although
most of the new leviviral genomes are partial, ~80
are either complete or nearly so, with all three core
genes annotated. Only one (AVE017) of these
~200 genomes had an annotated Sgl gene, being a
close relative (38% sequence identity) of MS2
L(85). Given their small size, predilection for being
embedded in the core genes, and extreme sequence
diversity at the protein level, finding Sgl candidates
in these genomes poses unique challenges to the
traditional gene annotation tools. Moreover,
currently there is no direct way to sort out these
leviviral genomes to particular bacterial host, or
more specifically, to a particular retractable pilus.
Nevertheless, the promise of more intriguing Sgl
proteins targeting novel components of the bacterial
cell wall machinery surely makes our current effort,
which involves identifying potential Sgl ORFs and
characterizing them one by one for the ability to
support lysis after induction of synthetic clones,
worth doing.
ACKNOWLEDGEMENTS
This work was supported by Public Health Service grant GM27099 and by the Center for Phage Technology
at Texas A&M University, jointly sponsored by Texas A&M AgriLife. The content is solely the
responsibility of the authors and does not necessarily represent the official views of the National Institutes
of Health. The following figures and figure legends were reproduced verbatim from published references:
Fig. 1, Fig. 3, Fig. 4, Supplementary figures 3, 4, 5, and 6. We thank past and current members of the Young
laboratory for their advice. We also thank Ing-Nang Wang, Kaspar Tars and Rene Olsthoorn for providing
ssRNA phage resources and expertise. The clerical assistance of Daisy Wilbert is highly appreciated.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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FIGURE LEGENDS
Figure 1. The PG biosynthesis pathway and the Sgl system.
(A) The PG precursor pathway, from cytoplasmic UDP-GlcNAc to periplasmic lipid II, with known targets
of 'protein antibiotics' indicated. (B) Genome organization in ssDNA ( X174) and ssRNA (Qβ, AP205,
MS2, PhiCb5, and M) phages. In ssRNA phages, mat encodes the maturation protein responsible for
adsorption to the receptor pilus, coat encodes the capsid protein, and rep encodes the replicase. In Qβ, the
mat gene is named A2 and has the additional function of inducing host lysis.
Figure 2. The E-resistant E. coli mraY alleles mapped on Aquifex aeolicus MraYAA.
The E. coli mraY alleles resistant to E are mapped on the structure of MraY from Aquifex aeolicus .(MraYAA)
(PDB 4J72). The structure is shown from the face that forms the putative E-MraY interface and the domains
of the interface are colored as follows; the periplasmic beta hairpin (yellow), the loop connecting PB with
TMD5 (purple), TMD5 (magenta), and TMD9 (9a and 9b) (grey). The homologous E-resistant residues in
MraYAA are shown as spheres and the catalytically important aspartate (red) and histidine (blue) residues
are shown as stick projections.
Figure 3. The structure of Qβ bound to MurA
(A) The structure of Qβ bound to MurA and it is colored as follows coat proteins (blue), A2 (hot pink),
gRNA (yellow) and MurA (orange). (B) A 90° turn and cutaway view of Qβ shows MurA bound to the
maturation protein with same color scheme as (A) except in case of coat proteins (radially colored from
light blue to blue) and extra protein density (green). (C) The ribbon model of A2 bound to MurA with
uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) in the active site (cornflower blue). (D) The
ribbon model of MurA viewed from the MurA–A2 interface. The point mutations that render MurA resistant
to A2 are labeled and shown as red stick models. Locations of the catalytic loop and the UDP-GlcNAc are
indicated by black arrows. (E) Ribbon model of A2 as viewed from the MurA–A2 interface. The region
interacting with MurA, encompassing the N-terminal β-sheet region of residues 30–120, is outlined by a
black lasso. The N and C termini are indicated by black arrows.
Figure 4. LysM-resistance mutations map to TMD2 and TMD7 of MurJ.
The amino-acid changes in E. coli MurJ (MurJEC) resulting in LysM-resistance were mapped onto the
structure of MurJ from Thermosipho africanus (MurJTA) (PDB 5T77). (A) The cytoplasmic-open
conformation. (B), A model of the periplasmic-open conformation (19). The TMDs that line the central
hydrophilic cavity are colored: TMD2 (light blue) and TMD7 (magenta). Lateral view (left) and periplasmic
view (right). The changes in MurJEC and homologous amino acids in MurJTA are shown on the right.
at Texas A&M University - Medical Sciences Library on November 12, 2018http://www.jbc.org/Downloaded from
Karthik Chamakura and Ry Young
Phage single-gene lysis: Finding the weak spot in the bacterial cell wall
published online November 12, 2018J. Biol. Chem.
10.1074/jbc.TM118.001773Access the most updated version of this article at doi:
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