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Solution and electron-microscopy characterization of lactococcal phage
baseplates expressed in E. coli.
Valérie Campanaccia, *, David Veeslera, Julie Lichièrea, Stéphanie Blangya, Giuliano Sciaraa,
Sylvain Moineaub, c, Douwe van Sinderend, e, Patrick Bronf and Christian Cambillaua, *
a Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 6098 CNRS and
Universités Aix-Marseille I & II, Campus de Luminy, Case 932, 13288 Marseille Cedex 09,
France
b Groupe de Recherche en Écologie Buccale (GREB) and Félix d’Hérelle Reference Center
for Bacterial Viruses, Faculté de Médecine Dentaire, Université Laval, Québec City, Québec,
Canada, G1V 0A6
c Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie,
Université Laval, Québec City, Québec, Canada, G1V 0A6
d Department of Microbiology, National University of Ireland, Cork, Ireland
e Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland
f Centre de Biochimie Structurale, INSERM U554 / CNRS UMR 5048, 29 rue de Navacelles,
34090 Montpellier, France
*Corresponding authors.
Fax: +33 491 266 720.
E-mail: [email protected] or [email protected].
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ABSTRACT
We report here the characterization of several large structural protein complexes
forming the baseplates (or part of them) of Siphoviridae phages infecting Lactococcus
lactis: TP901-1, Tuc2009 and p2. We revisited a “block cloning” expression strategy and
extended this approach to genomic fragments encoding proteins whose interacting
partners have not yet been clearly identified. Biophysical characterization of some of
these complexes using circular dichroism and size exclusion chromatography, coupled
with on-line light scattering and refractometry, demonstrated that the overproduced
recombinant proteins interact with each other to form large (up to 1.9 MDa) and stable
baseplate assemblies. Some of these complexes were characterized by electron
microscopy confirming their structural homogeneity as well as providing a picture of
their overall molecular shapes and symmetry. Finally, using these results, we were able
to highlight similarities and differences with the well characterized much larger
baseplate of the myophage T4.
Keywords: operon expression; Lactococcus lactis phage; multi-protein complex; multi-angle
light scattering; receptor binding protein; baseplate; electron microscopy.
Abbreviations: RBS, ribosome binding site; TMP, tape measure protein; Dit, distal tail
protein; Tal, tail-associated lysin; BppU, baseplate upper protein; BppL, baseplate lower
protein; RBP, receptor binding protein; SEC/MALS/RI, size exclusion chromatography
coupled with on-line static light scattering and refractometry; CD, circular dichroism; EM,
electron microscopy; TEV, tobacco etch virus.
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1. Introduction
During the course of several structural genomics programs, we designed several
methods to easily express hundreds of single proteins in Escherichia coli and screen for the
best conditions in order to obtain them in a soluble form and prone to crystallization (Alzari et
al., 2006; Campanacci et al., 2003; Graslund et al., 2008; Vincentelli et al., 2005; Vincentelli
et al., 2003; Vincentelli et al., 2004). However, the over-expression of individual subunits of a
multi-protein complex in E. coli can result in inclusion body formation, improper folding,
degraded products or cell growth inhibition due to product toxicity. In contrast, co-expression
of two or more subunits of such complexes can enhance expression yield, solubility, correct
folding and result in higher activities (Buddha et al., 2004; Dokland et al., 2002; Li et al.,
1997; Stebbins et al., 1999; Strong et al., 2006). Different strategies can be applied for co-
expression in E. coli such as the use of several vectors, a single vector with a single promoter,
a single vector with different promoters, or a combination of all of these approaches (for
review, see (Perrakis and Romier, 2008)). Recently, co-expression of four genes coding for
the Type IV secretion system made it possible to crystallize it and solve its 3D structure
(Chandran et al., 2009).
In this contribution, we describe the expression and characterization of the overall
assembly of different baseplates or baseplate components belonging to three lactococcal
phages (TP901-1, Tuc2009 and p2) infecting different strains of the low GC content gram-
positive lactic acid bacterium Lactococcus lactis (Fig. 1). Gene expression of lactococcal
phages is tightly and timely controlled during the replication cycle. In several cases, it was
shown that such phage genes are organized in at least three sequential clusters that are
temporally transcribed. The early genes are expressed just after infection and encode proteins
involved in phage replication and host control; the middle genes specify proteins involved in
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DNA packaging and morphogenesis; and the late genes mostly encode proteins allowing final
virion assembly and bacterial host lysis (Duplessis et al., 2005; Madsen and Hammer, 1998).
Besides, phage genomes are densely packed as more than 90% of the complete sequence
correspond to coding sequences (Hatfull, 2008). Each operon often contains clustered genes
having limited distance between each other. For example, in the L. lactis-infecting phage
TP901-1, genes that encompass orf37 through to orf41 all overlap, whereas for orf41 through
to orf49 (bppL) the distance between the stop codon of one gene and the ATG codon of the
following gene varies between 1 and 55 bp, with four non-coding intergenic regions of 12±3
bp (Brondsted et al., 2001; Johnsen et al., 1996). Similar densely packed genomes are also
observed in other lactococcal phages (Villion et al., 2009) including Tuc2009 (Seegers et al.,
2004).
Except for the production of receptor binding proteins (RBPs) (Ricagno et al., 2006;
Spinelli et al., 2006a; Spinelli et al., 2006b; Tremblay et al., 2006), our initial attempts to
express the individual components of lactococcal phages and reassemble them in huge
complexes failed in a large extent, due to lack of expression, inclusion body formation,
protein instability or the absence of interactions. We therefore implemented a partial operon-
based cloning strategy using a whole phage genomic segment that codes for two or more
phage structural proteins, followed by over-expression in E. coli. This strategy yielded
excellent expression levels of structural protein complexes from lactoccocal phages suitable
for biophysical and structural studies. This approach made it possible to obtain the baseplates
from phages TP901-1 (ORF46, ORF48 and ORF49) and p2 (ORF15, ORF16 and ORF18).
Moreover, we were able to produce chimerical baseplate protein complexes using a protein-
shuffling approach between the two related phages TP901-1 and Tuc2009. We then
characterized both individual proteins (when available) and complexes by circular dichroism
(CD) and SEC/MALS/RI (Veesler et al., 2009a; Veesler et al., 2009b). Some of these
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complexes were also studied by electron microscopy (EM) providing a picture of their overall
molecular shapes and symmetry. Finally, we used these results to compare lactococcal phage
baseplates organization to the well-known Myoviridae phage T4.
2. Materials and methods
2.1. Cloning procedure
All primers used were salt-free and purchased from Eurofins MWG Operon. Primers
were designed to introduce protein purification tags as well as att recombination sequences to
facilitate cloning. In silico optimized primers were designed using the ExSenSo software
(Care et al., 2008) and are listed in Table 1. DNA regions containing two or more genes (Fig.
1B) were amplified from genomic DNA using PrimeStar HS DNA polymerase (Takara Bio
Inc.) or Platinum Pfx DNA polymerase (Invitrogen) following manufacturers’ instructions.
For cloning of chimera or multiple, non-contiguous genes (to form an artificial operon, Fig.
1B), we amplified the individual genes with inclusion of an overlapping sequence before
performing a second PCR reaction to achieve joining of the initial PCR products. PCR
amplicons were cloned by Gateway recombination (Invitrogen) in pDEST™14 (Invitrogen),
pDEST™17 (Invitrogen) or pETG-20A (a kind gift from Dr Arie Geerlof, EMBL Hamburg)
vector, as previously described (Vincentelli et al., 2003). The various constructs are listed in
Table 2. All clones were confirmed by sequencing (GATC Biotech).
2.2. Protein production
Recombinant plasmids were transformed in E. coli Rosetta(DE3)pLysS (Novagen) or
T7 Express Iq pLysS (New England Biolabs) strains. Cells were grown at 37°C in Terrific
Broth or M9 minimal medium until the OD600nm reached 0.6, after which protein expression
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was induced with 0.5 mM IPTG overnight at 25°C or 17°C. Protein purification was
performed as previously described (Sciara et al., 2008; Siponen et al., 2009). Briefly, after
cell harvesting, lysis was performed by adding 0.25 mg/ml lysozyme, followed by a
freezing/thawing cycle and sonication. Soluble proteins were separated from inclusion bodies
and cell debris by a 30-min centrifugation step at 20,000 x g. Purification was performed on
an ÄKTA FPLC system in two steps: a Ni2+-NTA column (HisTrap Ni 5 mL, GE Healthcare)
with a step gradient of 250 mM imidazole, followed by a preparative Superdex 200 HR 26/60
gel filtration. For complexes with a Strep-tag II, an additional affinity chromatography was
performed prior to gel filtration by means of a Strep-Tactin superflow resin (IBA GmbH)
using 2.5 mM D-desthiobiotin as eluant. For the thioredoxin fusion, a cleavage by
recombinant TEV protease was performed after the Ni affinity chromatography.
2.3. Determination of complexes molecular masses by SEC/MALS/RI
Size exclusion chromatography was carried out on an Alliance 2695 HPLC system
(Waters) using either a Superose 6 HR10/30 column (GE Healthcare) or a KW405-4F column
(Shodex) run in a buffer containing 10 mM Hepes, 150 mM NaCl, and 0.02% NaN3 at pH 7.5
with a flow rate of 0.3 mL/min or 0.35 mL/min, respectively. Detection was performed using
a three-detectors static light-scattering apparatus (MiniDAWN TREOS, Wyatt Technology), a
quasi-elastic light-scattering instrument (Dynapro, Wyatt Technology) and a refractometer
(Optilab rEX, Wyatt Technology). Molecular weight calculations were performed with the
ASTRA V software (Wyatt Technology) using a dn/dc value of 0.185 mL/g, as previously
described (Veesler et al., 2009a). Proteins were injected at a final concentration of 0.02 mM.
Errors were assigned by the Astra software (Wyatt).
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2.4. Circular dichroism
Far-UV CD spectra were recorded with a JASCO J-810 spectropolarimeter (JASCO
Corporation) equipped with a Peltier temperature control and using 1-mm thick quartz cells.
CD spectra were averaged on three accumulations using a scanning speed of 50 nm/min.
Measurements were performed between 190 and 260 nm at 20°C in 10 mM Na/Na2 phosphate
buffer pH 7.5 with a protein concentration of 0.1 mg/mL. Mean residual ellipticity values
([Θ]) were calculated as [Θ] = 3300 M ∆A/(l•c•n), where l (path length) = 0.1 cm, n = number
of residues, M = molecular mass (Daltons), and c = protein concentration (mg/mL).
Secondary structure analysis was done with the Dichroweb server (Whitmore and Wallace,
2004) using the CDSSTR method (Sreerama and Woody, 2000) and the reference set SP175
(Lees et al., 2006).
2.5. Electron microscopy and image processing
Samples were diluted to a final concentration of 0.05 mg/mL. Three microliters of a
given protein sample was applied to glow-discharged, carbon-coated, copper grids. Following
a 2 min exposure period to allow contact, fluid excess was removed by blotting paper and 4 µl
of 1% uranyl-acetate were added to the grid and left for 1 min. The grids were then dried and
kept in a desiccator cabinet until examination. Electron micrographs were recorded under
low-dose conditions with a FEI Tecnai Sphera LaB6 200 kV microscope. Images were
collected at 50,000X and 29,000X magnification for TP901-1 complexes and p2 baseplate,
respectively; defocus range was of 0.4 to 1.0 µm. Micrographs were checked by optical
diffraction and digitized on a Nikon Coolscan 9000 ED with a step size of 10 µm. The
digitized images were coarsened by a factor of 2, resulting in a pixel size corresponding to 4
Å and 6.9 Å at the specimen level for the TP901-1 and p2 complexes, respectively.
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Preliminary image processing was performed using the IMAGIC V software (van Heel
et al., 1996). Briefly, single molecule images were extracted semi-automatically from raw
micrographs using Boxer (Ludtke et al., 1999). The phase-contrast-transfer function was
corrected by phase flipping. Images were auto-centered employing the CENTER-IMAGE
program by using the average of raw images as reference. Some preferential views were
selected by visual inspection and chosen as references using the MRA (multi reference
alignment) program from the IMAGIC V software. Images were then grouped into classes
and averaged using the MSA (multi-statistical alignment) procedure. Best images of each
averaged class were used as new references for a new alignment cycle. In the present study,
three iterative alignment cycles of images were performed.
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3. Results
3.1. Production of baseplate proteins from lactococcal phages TP901-1 and Tuc2009.
TP901-1 and Tuc2009 are two temperate phages belonging to the lactococcal
Siphoviridae P335 group (Deveau et al., 2006; Vegge et al., 2006). The distal tail structure of
TP901-1 contains an initiator complex formed by the tape measure protein (TMP, ORF45),
the distal tail protein (Dit, ORF46) and the tail-associated lysin (Tal, ORF47) as well as a
double-disk-shaped baseplate, which is made of a lower baseplate protein (BppL also called
receptor binding protein (RBP), ORF49) and an upper baseplate protein (BppU, ORF48)
(Vegge et al., 2005; Vegge et al., 2006). BppL was previously shown to be involved in the
host recognition process (Vegge et al., 2006).
We individually over-produced and purified TP901-1 BppU and BppL yielding 4 and
2 mg of pure protein per liter of culture, respectively (Table 2, rows 1 & 2). As we observed
that mixing of BppU and BppL from Tuc2009 did not result in complex formation (Sciara et
al., 2008), we generated bicistronic vectors for the co-expression of TP901-1 and Tuc2009
bppU/bppL to overcome this problem. In these constructs, an in silico optimized ribosome
binding site sequence (RBS) was introduced to promote efficient translation of bppU and
bppL transcripts (Care et al., 2008). Furthermore, constructs were designed in such a way that
BppU and BppL carried a hexahistidine tag and a Strep-tag II (Schmidt and Skerra, 2007),
respectively (constructs 3 and 11 in Figure 1B). For TP901-1, we did not observe the
expression of a protein complex (Table 2, row 3). In contrast, clear production of a
BppU/BppL complex was observed for the Tuc2009-derived proteins, enabling us to
previously propose a topological model for the baseplate assembly of this latter phage (Sciara
et al., 2008).
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Because operon-based expression has been successfully used in some cases (Droge et
al., 2000; Fronzes et al., 2009; Takagi et al., 2005; Wang et al., 2006; Wilbur et al., 2005), we
decided to clone a larger phage genomic fragment containing the genes coding for BppU and
BppL from TP901-1 or Tuc2009 as well as the intervening sequence between these two
genes. Moreover, to test the influence of RBS sequences, two constructs were designed where
either the endogenous RBS or an in silico optimized RBS (Care et al., 2008) controlled bppU
mRNA translation, while endogenous lactococcal phage RBS of bppL and bppA (a baseplate-
associated protein found in Tuc2009) were kept (Tables 1, 2). A hexahistidine tag was
introduced at the BppL C-terminus for purification purpose.
For the TP901-1 baseplate proteins, excellent expression levels were achieved using
an in silico optimized RBS upstream of bppU: 10 mg of the BppU/BppL complex and 20 mg
of uncomplexed excess BppL were obtained per liter of culture (Table 2, row 4). This latter
uncomplexed protein was not subject to proteolytic cleavage, in contrast to what was
previously observed (Spinelli et al., 2006a). This might result from a more favorable
BppL:protease ratio as compared to the situation where BppL was expressed alone. It is
noteworthy to point out that the TP901-1 BppU/BppL complex production dropped
considerably when the endogenous RBS of bppU was used (Table 2, row 5). Likewise, we
designed an artificial operon containing the genes encoding Dit, BppU and BppL and cloned
it in frame as a C-terminal fusion to the thioredoxin gene. After two affinity-purification steps
(including a TEV protease cleavage) and a gel filtration, we obtained 8 mg per liter of culture
of a stable complex constituted by these three proteins as well as an excess of Dit (Table 2,
row 6).
Surprisingly, for the Tuc2009 baseplate proteins, no BppU/BppL complex was
produced using either an endogenous or an in silico optimized RBS upstream of bppU,
although we obtained large amounts of BppL alone (Table 2, rows 12 & 13). We observed the
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BppU/BppA/BppL complex formation in low amounts (Table 2, row 14), but its co-elution
with the host chaperone protein DnaK during gel filtration (data not shown) precluded further
characterization.
We were thus able to produce and purify individual baseplate proteins from the two
P335 lactococcal phages, TP901-1 and Tuc2009. Furthermore, our operon-based approach
coupled with optimal RBS screening allowed us to obtain high quantity of several protein
complexes formed by two or three protein components.
3.2. Production of chimerical baseplate complexes from TP901-1 and Tuc2009 lactococcal
phages
Isolation of chimerical phages or phage mutants was originally used with the RBP-
encoding gene in E. coli T-even phages (Tetart et al., 1998; Tetart et al., 1996). This approach
was also successfully used for Streptococcus thermophilus phages (Duplessis and Moineau,
2001), in two Lactobacillus delbrueckii infecting phages (Ravin et al., 2002) and in several
lactococcal phages (Dupont et al., 2004; Stuer-Lauridsen et al., 2003; Vegge et al., 2006). In
the latter case, it was shown that a chimerical TP901-1 phage encoding the BppL from
Tuc2009 (BppLTuc2009) exhibited altered host range specificity (Vegge et al., 2006). We thus
decided to produce both BppUTP901-1/BppLTuc2009 and BppUTuc2009/BppLTP901-1 chimerical
complexes. Using the strategy described above for the TP901-1 BppU/BppL complex, we
cloned a DNA fragment containing the bppUTP901-1 (or bppUTuc2009) gene with an optimized
RBS upstream, and, the bppLTuc2009 (or bppLTP901-1) gene with the endogenous bppLTP901-1
RBS. As for the TP901-1 complex, high yields of purified complexes were obtained (5 and 15
mg/L for BppUTP901-1/BppLTuc2009 and BppUTuc2009/BppLTP901-1 respectively), as well as a
large excess of either BppLTuc2009 or BppLTP901-1 (Table 2, rows 15 & 16).
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3.3. Production of baseplate proteins from the lactococcal phage p2
The virulent phage p2 belongs to the lactococcal 936 group, which represents the
predominant phage group responsible for industrial milk fermentation failures (Chandry et al.,
1997; Crutz-Le Coq et al., 2002; Mahony et al., 2006). We previously demonstrated that
ORF18 of phage p2 is located within the baseplate and identified it as the RBP (Spinelli et al.,
2006b). Furthermore, ORF14 of phage p2 is most likely the tape measure protein (TMP)
based on its genomic position and on the fact that it is the largest structural protein (Dupont et
al., 2004). Similarly, using genomic organization as well as gene sequence similarities (albeit
very limited) with their homologous counterparts in phages TP901-1 and Tuc2009, we
hypothesized that the p2 phage baseplate is encoded by orf15 to orf18.
The DNA fragment encompassing orf15 through to orf18 was PCR-amplified using
primers designed to add a hexahistidine tag at the N-terminus of ORF15 and a Strep-tag II at
the C-terminus of ORF18. Following two different affinity chromatography steps and a gel
filtration, we obtained a high yield of the ORF15/ORF16/ORF18 complex (6 mg per liter of
culture) as well as some excess ORF15/ORF18 complex (Table 2, row 17). ORF17 was
neither detectable associated with any of these two complexes nor in the whole cell lysate.
This is in agreement with its absence in virion particles (data not shown) and suggests a non-
structural role for this protein.
To further investigate the interaction network, we also cloned orf15 and orf16, using
our operon strategy, and obtained 4 mg per liter of culture of the ORF15/ORF16 complex
(Table 2, row 18). As stable complexes of ORF15/ORF16, ORF15/ORF18 and
ORF15/ORF16/ORF18 could be obtained, we hypothesize that ORF15 plays a central
structural role in the p2 baseplate assembly, as was proposed for the Dit protein in the P335-
group of lactococcal phages (Vegge et al., 2005; Vegge et al., 2006).
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3.4. Molecular mass and size determination of baseplate protein complexes
SEC/MALS/RI characterization (Veesler et al., 2009a) (Table 2, Fig. 2) revealed
masses of 102,000 Da and 54,000 Da for TP901-1 BppU and BppL, respectively (Table 2,
rows 1 & 4; Fig. 2A). These data attest the trimeric nature of both proteins. Analysis of the
TP901-1 BppU/BppL complex resulted in a measured mass of 255,000 Da, suggesting a 3:9
ratio (Table 2, row 4; Fig. 2A), that is compatible with the observation of a tripod in EM (see
below). Interestingly, this mass was identical to the one obtained for the Tuc2009
BppU/BppA/BppL complex (Table 2, row 14) (Sciara et al., 2008) indicating that, in this
latter phage, 6 BppL have been replaced by 3 BppA, the exact role of this latter protein
remaining still undocumented. The measured molar mass of the Dit/BppU/BppL complex was
1,900,000 Da. Combining this latter result with the EM evidence of a 6-fold symmetry (see
below), strongly suggests the presence of 6 (BppU/BppL) tripods associated with 12 Dit,
yielding a 12:18:54 ratio of Dit/BppU/BppL. However, it should be noted that expressing Dit
alone surprisingly resulted in a monomer (27,300 Da) (Table 2, row 6). This multimerization
process upon partner binding is a very common feature in phages as illustrated by the phage
λ tail terminator protein (gpU) (Pell et al., 2009), the phage λ capsid-stabilizing protein (gpD)
(Imber et al., 1980; Yang et al., 2000), the Bacillus subtilis SPP1 phage adaptor (gp15) and
stopper (gp16) proteins (Lhuillier et al., 2009) as well as the phage P22 tail accessory factor
(gp4) (Olia et al., 2006).
Analysis of the BppUTP901-1/BppLTuc2009 and BppUTuc2009/BppLTP901-1 chimeras
provided complexes with 3:9 ratios deduced from measured masses of 264,000 Da and
284,000 Da, respectively (Table 2, rows 15 & 16). As observed for the Tuc2009 proteins
(Sciara et al., 2008), higher molecular weight complexes, corresponding to 2x(3:9) and
3x(3:9) oligomers, were also detected for these two chimeras. These results suggest that, in a
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chimerical context with TP901-1, either BppUTuc2009 or BppLTuc2009 proteins assemble as a
native TP901-1 BppU/BppL complex with respect to their stoichiometry.
Complex formation was also observed when mixing BppLTP901-1, obtained from this
operon-based production, with individually produced BppUTP901-1 or BppUTuc2009, (Table 2,
row 7; Fig. 2A). In contrast, no complex formation occurred when BppLTuc2009 was added to
BppUTP901-1 or BppUTuc2009 (data not shown). This is consistent with our previous
observations using separately produced Tuc2009 proteins (Sciara et al., 2008) and is probably
due to the rapid proteolytic degradation of BppL, as illustrated by crystallographic structures
(Sciara et al., 2008; Siponen et al., 2009; Spinelli et al., 2006a). This indicates a full integrity
requirement of the BppL N-terminus for protein complex formation.
Analyses of the p2 protein complexes revealed masses of approximately 1,000,000
Da, 120,000 Da and 310,000 Da for ORF15/ORF16/ORF18, ORF15/ORF18 and
ORF15/ORF16, respectively (Table 2, rows 17 & 18; Fig. 2B). As ORF18 is a trimer (Spinelli
et al., 2006b; Tremblay et al., 2006), we thus propose that the ORF15/ORF18 complex exists
in a 1:3 protein ratio, the ORF15/ORF16 complex in a 6:3 ratio, while the
ORF15/ORF16/ORF18 complex, considering its 6-fold symmetry seen by EM (see below),
might occur in ratios of 12:3:18 or 6:3:18, of respective masses 1,100,000 and 900,000 Da.
3.5. Characterization of TP901-1 and chimerical BppU/BppL complexes by circular
dichroism
In order to verify the structural integrity of the different BppU/BppL complexes, far-
UV circular dichroism spectra were recorded. All spectra were typical of structured proteins
(data not shown). Estimation of secondary structural contents based on CD spectra
deconvolution revealed a significant discrepancy in the α-helical and β-strand contents
between the complex from TP901-1 (7% α-helix and 43% β-strands) and the two chimeras
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(17%-18% α-helix and 37% β-strand, Table 3). These results associated with the
SEC/MALS/RI ones, suggest that BppU and BppL shuffling between P335-like phages can
lead to quite stable complexes, thereby illustrating means by which these phages evolve
(Haggard-Ljungquist et al., 1992; Labrie et al., 2008). Obtaining such shuffled complexes
also reinforces our hypothesis that the BppL N-terminus mediates anchoring to BppU because
of the observed 64% amino acid identity between the first 20 residues of the two BppL
proteins (from Tuc2009 and TP901-1 phages) (Mc Grath et al., 2006). Finally, the secondary
structural content discrepancies between chimeras and native TP901-1 complex may be
explained by the presence of BppA in the native Tuc2009 baseplate as an additional partner
during protein complex formation.
3.6. Electron microscopy of protein complexes reveals baseplate structures
The over-expressed TP901-1 Bppu/BppL and Dit/BppU/BppL as well as p2
ORF15/ORF16/ORF18 complexes, were negatively stained using 1% uranyl-acetate and
observed with a transmission electron microscope (Fig. 3). Particle images were extracted
from digitized images and processed using the IMAGIC V software. Images were centered,
aligned, and grouped into classes presenting structural similarities. We selected representative
class averages (insets in Fig. 3A) computed after 3 alignment cycles of raw images. In the
case of the TP901-1 Bppu/BppL complex, particles were adsorbed on the carbon film and
well resolved. Many of them appear to have a fork-like shape showing two or three prongs
(tripod, Fig. 3A, indicated by black arrows) terminated by a globular domain. Particles are
approximately 16.4 nm long and 13.6 nm wide. It is worth noting that most class averages
were related to these structural characteristics highlighting the structural homogeneity of
BppU/BppL particles.
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The production of TP901-1 Dit/BppU/BppL complex (without Tal) resulted in the
formation of particles displaying protuberances at the periphery and annular densities in the
middle, as illustrated in Fig. 3B. Image analysis revealed two main class averages presented
in insets of Fig. 3B. These two averaged views are similar but not identical, corresponding
probably to the top and bottom view of these particles. They reveal an apparent 6-fold
symmetry axis with two central rings connected via small-elongated parts to protruding
triangular domains. Outer diameters of the whole particle and of the inner ring are 45 nm and
13.4 nm, respectively. The 10 nm long protruding domains are linked to the external ring
consisting of 12 circular densities being approximately 3.4 nm wide.
In the case of the ORF15/ORF16/ORF18 protein complex from phage p2,
micrographs analysis reveals particles with a highly homogeneous circular shape. Image
processing indicated that particles had preferential views showing an apparent 6-fold
symmetry axis with two rings of density containing well resolved domains inside, as shown in
the class averages (Fig. 3C, insets). The two ring outer diameters are 23.5 nm and 9.6 nm,
respectively. Most of the identified class averages correspond to those shown in the insets 1
and 2 of Fig. 3C. This is typical of a particle interacting in a preferential way with the carbon
film of the grid. Class averages 1 and 2 of Fig. 3C possibly correspond to the up and down
view, while class average 3 is a presumed representation of the side view of the particle.
3. Discussion
We obtained different baseplate protein complexes, belonging to lactococcal phages
TP901-1, Tuc2009, and p2, thanks to an expression strategy in which internal translational
signals were conserved using operon fragments encompassing 2 to 4 different genes. Within
this context, this approach is both faster and more efficient than those involving classical
multi-cistronic vectors and was useful to determine interacting partners. We were also able to
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obtain chimerical complexes where the BppL of two phages belonging to the P335 group
were shuffled providing an interesting approach to study phage evolution.
Over-expression of three complexes, TP901-1 BppU/BppL and Dit/BppU/BppL as
well as p2 ORFs 15/16/18, yielded particles showing relative homogeneity as revealed by the
well-resolved EM class averages. Although these complexes interact with the carbon film of
the EM grid, limiting thus the number of observable orientations, their overall shape and
symmetry can be deduced from both SEC/MALS/RI and EM results. This approach can be
applied to phages with relatively small baseplates such as Lactococcus lactis siphophages. It
would be probably more difficult or even impossible for myophages, such as T4, which are
much larger and composed of numerous different ORFs.
EM pictures of TP901-1 Bppu/BppL particles reveal that they might be composed of
two or three elongated domains. Combining these results with our SEC/MALS/RI analysis,
led us to propose that the tripod architecture is the most probable one, in which a trimer of
BppU forms the dome-shaped region of the tripod, while BppL trimers form each foot. In the
Dit/BppU/BppL complex, Dit most probably form a dodecameric hub structure (indicated by
the SEC/MALS/RI data) on which six tripods would be accommodated, leading to the overall
6 fold symmetry revealed by the EM pictures. The formation of such a complex constituted
by 12 Dit, 18 BppU and 54 BppL (Table 2) allows us to rule out that Tal, a component of the
initiator complex, is an essential element in the initial baseplate assembly of P335 phages.
Vegge et al. showed that tal- or dit- mutant phages exhibited head structures only, lacking the
entire tail (Vegge et al., 2005). Our results clearly demonstrated that the lack of Tal did not
prevent the formation of a stable TP901-1 baseplate structure. We therefore hypothesize that
Tal, or at least its N-terminal part, exerts a role in anchoring the baseplate to the tail,
stabilizing thus the phage tail probably via interactions with the TMP (Mc Grath et al., 2006).
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We identified the three proteins forming the p2 baseplate, namely ORF15, ORF16 and
ORF18. This complex has an apparent mass of ~1,0 MDa, that is in between masses of
complexes having 6 or 12 ORF15, 3 ORF16 and 18 ORF18 (Table 2). If 12 ORF15 were
present, the assembly of p2 ORF15 would be comparable to that of TP901-1 Dit. The role of
ORF15 as a hub on which other proteins are plugged is consistent with the presence of the
isolated ORF16/ORF18 complex having a 1:3 stoichiometry (Table 2). Furthermore, the fact
that ORF16 is trimeric could indicate an analogous function to Tal proteins in P335 phages
(Sciara et al., 2008). The transition from 6 to 3-fold symmetry in baseplates is a feature which
has also been reported in the myophage T4 (Kanamaru et al., 2002).
It is noteworthy that an overall 6-fold symmetry is exhibited by both baseplates, from
TP901-1 and p2 phages. The same symmetry was also observed for the baseplate of the
myophage T4 despite its larger mass (accounted by ~130 proteins of 14 different types) and
size (520 nm large x 270 nm high) (Leiman et al., 2004; Rossmann et al., 2004). In this latter
case, the 6-fold to 3-fold symmetry transition is observed at the level of the puncturing
device. This should be also observed with phage p2 baseplate (3 ORFs16) and is very likely
the case with phage TP901-1 Tal (as is the case in Tuc2009).
Myoviridae phages as well as some Siphoviridae phages possess a baseplate for
adsorption to the host cell wall. This feature seems to be the hallmark of phages binding to
saccharidic receptors. It is remarkable that both phage families appear to have convergently
evolved to adopt such complex structures. This work, dedicated to lactococcal phage
baseplates characterization, makes it possible to pursue higher resolution analysis of these
large complexes, with the perspective to understand mechanisms taking place during infection
at the molecular level.
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Acknowledgements
We thank Dr. Arie Geerlof, who kindly provided the Gateway plasmid pETG-20A for
His-Trx fusion. This work was supported, in part, by grants from the Marseille-Nice
Génopole (to J.L.), the CNRS, the Natural Sciences and Engineering Research Council of
Canada through its strategic program (to S.M.), the Science Foundation Ireland Principal
Investigator program (ref. no. 08/IN.1/B1909 (to D.v.S.)), the ANR (ANR-07-BLAN-0095)
and by a PhD grant from the "Ministère français de l'Enseignement Supérieur et de la
Recherche" to D.V. (ref. no. 22976-2006).
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Figure legends
Fig. 1. Schematic representation of the orfs constituting the baseplate of the lactococcal
phages TP901-1, Tuc2009 and p2 and the expression constructs of their complexes. (A) The
structural genes encompassing the orfs belonging to the segment following the TMP and
finishing at the RBP. (B) Schematic view of the various constructs generated during this
study. The numbers in parenthesis correspond to the lines in Table 2. The red, green and
orange flags represent His6 tag, Strep tag and TRX fusion, respectively. Full lines and dashed
lines represent endogenous and engineered RBS, respectively.
Fig. 2. SEC/MALS/RI analysis of the different proteins and complexes. The molar mass (left
axis, solid lines) and the UV280nm absorbance (right axis, dashed lines) are plotted as a
function of the column elution volume. (A) TP901-1 BppU (pink), BppL (blue), BppU/BppL
(green) and mix of individually produced BppU with BppL from operon expressed
BppU/BppL (black). The column used was a 24 mL Superose 6 HR10/30 column (GE
Healthcare). (B) TP901-1 Dit/BppU/BppL complex (green), p2 ORF15/18 (orange) and p2
ORF15/16/18 (violet). The column used was a 5 mL KW405-4F column (Shodex).
Fig. 3. Electron microscopy analysis and image processing of negatively stained images of
TP901-1 BppU/BppL and Dit/BppU/BppL complexes as well as p2 baseplate proteins.
Images of TP901-1 BppU/BppL complex (A), TP901-1 Dit/BppU/BppL complex (B) and p2
baseplate (C) were recorded at 50,000 X (A and B) and 29,000 X (C) magnification. Scale
bar, 500 Å. Protein complexes were observed using 1% uranyl-acetate and imaged on a FEI
Tecnai Sphera LaB6 200 kV microscope. Proteins are in white. The insets show representative
class averages obtained after three iterative alignment cycles of raw images. The size of insets
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in A, B and C are 360 Å x 360 Å, 983 Å x 983 Å and 621 Å x 621 Å, respectively. No
symmetry was applied on images.
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Table 1. Primers sequences and features
Name Sequence (5’ to 3’)a
TP901-1 BppU-ins-F attB1-GAAGGAGATAGAACC ATG AAA ACA GAA CAT TTT ATA ACA CTG TC
BppU-endo-F attB1-AGAAAGGGTCTATT ATG ACA GAA CAT TTT ATA ACA CTG TCC ACC
BppU-HC-R attB2-CTA gtg atg gtg atg gtg atg ATC TAT TGG ATA TGT ACC AGT C
BppU/L-ins-F cat cac cat cac cat cac TAA TAATCTAGAAATTTTGTTTAACTTTAAGAAGGAGATAGAACC ATG AAA GCT AGT ATT AAA AAA GTA TAT CGT GGC
BppU/L-ins-R TTT CAT GGTTCTATCTCCTTCTTAAAGTTAAACAAAATTTCTAGATTA TTA gtg atg gtg atg gtg atg AGC TAC AAA AAC ATA GCC TTT TG
BppL-ins-F attB1-GAAGGAGATAGAACC ATG GCT AGT ATT AAA AAA GTA TAT CGT GG
BppL-SC-R attB2-TTA TTA ttt ttc gaa ctg cgg gtg gct cca agc gct ATC TAT TGG ATA TGT ACC AGT CC
Dit-F attB1- TA gaa aac ctg tac ttc cag ggt TAC AAG TTT AGA GAT ACG AC
Dit/BppU-F CG ATT CAA TAT AGA AAG GCG GTA CTT TAA AAAAAGGAGAAAAA ATG ACA GAA CAT TTT ATA ACA CTG TCC ACC
Dit/BppU-R GGT GGA CAG TGT TAT AAA ATG TTC TGT CAT TTTTTCTCCTTTTT TTA AAG TAC CGC CTT TCT ATA TTG AAT CG
Tuc2009
BppU-ins-F attB1-GAAGGAGATAGAACC ATG AAA ACAGAACATTTTATAACACTGTCC
BppU-endo-F attB1-AGAAAGGGTCTATT ATG ACA GAA CAT TTT ATA ACA CTG TCC ACC
BppU/L-endo-F CG TGG GAA AAC GGG GGA TAA TAGAAATAGGAGAATAAA ATG GCT GAA TTA ACT AAA ATT AC
BppU/L-endo-R GT AAT TTT AGT TAA TTC AGC CAT TTTATTCTCCTATTTCTA TTA TCC CCC GTT TTC CCA CG
BppL-HC-R attB2-TTA gtg atg gtg atg gtg atg ATT CCG ATA AAG TTT TAC AAT C
Chimera
BppUTP901-1/LTuc2009-F ACA AAA GGC TAT GTT TTT GTA GCT TAA AAAAAGGAGAAAAA ATG GCT GAA TTA ACT AAA ATT ACT CG
BppUTP901-1/LTuc2009-R CG AGT AAT TTT AGT TAA TTC AGC CAT TTTTTCTCCTTTTT TTA AGC TAC AAA AAC ATA GCC TTT TGT
BppUTuc2009/LTP901-1-F TT TCG TGG GAA AAC GGG GGA TAA AAAAAGGAGAAAAA ATG GCT AGT ATT AAA AAA GTA TAT CGT GG
BppUTuc2009/LTP901-1-R CC ACG ATA TAC TTT TTT AAT ACT AGC CAT TTTTTCTCCTTTTT TTA TCC CCC GTT TTC CCA CGA AA
p2
ORF15-F attB1-TA gaa aac ctg tac ttc cag ggt GTA AGA CAG TAC AAA ATA CAT AC
ORF16-R attB2-TTA TTA
ORF18-R attB2-TTA TTA ttt ttc gaa ctg cgg gtg gct cca agc gct TTT AAT GAA GTA ACT TCC GTT ACC
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a For clarity, attB1 (5’-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3’) and attB2 (5’-GGGGACCACTTTGTACAAGAAAGCTGGGT-3’)
sequences are abbreviated in the table. RBS are underlined, ATG and stop codons in bold, tag encoding sequences in lower case, lysine codon in
italic and capitals, Tobacco Etch Virus (TEV) protease encoding sequence in italic and lower case.
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Table 2. Summary of protein production and SEC/MALS analysis on baseplate proteins and complexes
Name First
gene RBS
origin
Middle
gene
RBS
origin
Last
gene
RBS
origin
Purified proteins Purification
yield
(mg/L)
Theoretical
MW (kDa)
SEC/MALS MW
(in kDa) d
Protein
ratio
Measured RH (nm)
(hydrodynamic
radius)
TP901-1 1- BppU-His-C in silico - - BppU 4 34.8 102±0.10 3 4.27±0.27 2
-
BppL-His-C in silico - - BppL 2 18.0 - -
3
-
BppU-His-C/BppL-Strep-C
in silico in silico
- BppU/BppL & BppL
- 1
34.8 / 18.5 18.5
- - -
4
-
BppU/BppL-His-C in silico endo - BppU/BppL & BppL
10 20
34.0 / 18.0 18.0
255±0.20 54±0.40
3:9 3
6.0±0.38 3.42±0.22
5
-
BppU/BppL-His-C endo endo - BppU/BppL & BppL
2 10
34.0 / 18.0 18.0
- -
- -
-
6
-
Dit/BppU/BppL-Strep-C
pETG-20A
c
endo (bppL)
endo Dit/BppU/BppL & Dit
8 3
29.0 / 34.0 / 18.5 29.0
1,906±17.0 27.3±0.20
12:18:54 1
12.25±1.06 3.06±0.19
7
-
BppU-His-C + BppL-His-C
a
- - - - - 34.0 / 18.0
231.7±0.3 – 127.9±1.20 –
54.8±1.28
3:9 - 3 (BppU) - 3 (BppL)
-
Tuc2009
8
-
BppU-His-Cb in silico - - BppU 25 37.4 109.3±0.5 3 4.76±0.17
9
-
BppA-His-Cb in silico - - BppA 3 32.9 28.1±0.49 1 2.48±0.20
1
0-
BppL-His-Cb in silico - - BppL 12 20.0 60.3±0.23 3 -
1
1-
BppU-His-C/BppL-Strep-C
b
in silico in silico
- BppU/BppL & BppU
2 15
37.4 / 20.2 37.4
370±2.41 170 ±1.72
3:3 – 2x(3:3)
-
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1
2-
BppU/BppL-His-C endo endo - BppU/BppL & BppL
- 10
36.4 / 19.7 19.7
- -
- -
1
3-
BppU/BppL-His-C in silico endo - BppU/BppL & BppL
- 25
36.5 / 19.7 19.7
- -
- -
1
4-
BppU/BppA/BppL-His-C
b
in silico endo endo BppU/BppA/BppL & BppL
<1 25
36.5 / 31.9 / 19.7 19.7
255±1.43 -
3:3:3 -
Chimera BppU/BppL
1
5-
BppUTP901-
1/BppLTuc2009-His-C in silico Endo
(bppL
TP901-1)
- BppUTP901-
1/BppLTuc2009
& BppLTuc2009
5 40
34.0 / 18.0 19.7
494.8±2.50 - 264.8±0.5
2x(3:9) - 3:9
- -
1
6-
BppUTuc2009/BppLTP901-
1-His-C in silico endo
- BppUTuc2009/BppLTP901-
1
& BppLTP901-1
15 30
36.5 / 18.0 18.0
794.0±1.40 -581±1.2 -
284.0±3.10
3x(3:9) - 2x(3:9) -
3:9
- - -
p2
1
7-
ORF15-His-N/ORF16/ ORF17/ORF18-Strep-C
pDEST17d endo endo ORF15/ORF16/ORF18
& ORF15/ORF18
6
<1
37.8 / 42.8 / 29.8
37.9 / 29.8
1,004±150.0
132.2±0.50
6:3:18 or 12:3:18
1:3
-
4.7±0.25
1
8-
ORF15-His-N/ORF16 pDEST17d endo - ORF15/ORF16
& ORF15 4
<1 37.8 / 42.8 37.8
305.8±1.40 -
6:3 -
6.78±0.42
a individually produced BppU-His-C was mixed with excess BppL-His-C obtained via BppU/BppL-His-C operon expression;
b from (Sciara et al., 2008);
c RBS from the pETG-20A vector;
d RBS from the pDEST17 vector. ‘Endo’ and ‘in silico’ refer to endogenous and ‘in silico’ designed RBS, respectively.
When using the endogenous RBS from an another gene, its origin is indicated between parentheses. d
errors estimated for each experiment with the Astra software (Wyatt inc.). Note that the real MWs can be within 10% from the measured values.
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Table 3. Secondary structure content analysis obtained by circular dichroism of BppU/BppL
complexes
Helix1 Helix2 Strand1 Strand2 Turns Other NRMSDa
TP901-1 BppU/BppL
0.01 0.06 0.29 0.14 0.11 0.37 0.052
Chimera BppUTP901-1/BppLTuc2009
0.07 0.10 0.25 0.12 0.10 0.35 0.078
Chimera BppUTuc2009/BppLTP901-1
0.08 0.10 0.25 0.12 0.10 0.36 0.026
a NRMSD (normalized root-mean-square deviation) parameter (Mao et al., 1982) is a measure
of the difference between the experimental ellipticities ( exp) and the ellipticities of the back-
calculated spectra ( cal) for the derived structure. NRMSD is defined as [( exp-
cal)2/( exp)
2]1/2
, summed over all wavelengths. NRMSD values of <0.1 mean that the back-
calculated and experimental spectra are in close agreement (Brahms and Brahms, 1980).