Targeted recognition of Lactococcus lactis phages to polysaccharide receptors

The targeted recognition of Lactococcus lactis phages to
their polysaccharide receptors
Orla McCabe,1† Silvia Spinelli,2,3† Carine Farenc,2,3
Myriam Labbé,4,5 Denise Tremblay,4
Stéphanie Blangy,2,3 Stefan Oscarson,1*
Sylvain Moineau4,5 and Christian Cambillau2,3*
1Centre for Molecular Innovation and Drug Discovery,
School of Chemistry and Chemical Biology, University
College Dublin, Belfield, Dublin, Ireland.
2Architecture et Fonction des Macromolécules
Biologiques, CNRS, Marseille, UMR 7257, France.
3Aix-Marseille University, Campus de Luminy, Case
932, Marseille, 13288 France.
4Groupe de recherche en écologie buccale & Félix
d’Hérelle Reference Center for Bacterial Viruses,
Faculté de médecine dentaire, Université Laval,
Québec, G1V 0A6, Canada.
5Département de biochimie, de microbiologie et de
bio-informatique, Faculté des sciences et de génie,
Université Laval, Québec, G1V 0A6, Canada.
Summary
Each phage infects a limited number of bacterial
strains through highly specific interactions of the
receptor-binding protein (RBP) at the tip of phage tail
and the receptor at the bacterial surface. Lactococcus
lactis is covered with a thin polysaccharide pellicle
(hexasaccharide repeating units), which is used by a

subgroup of phages as a receptor. Using L. lactis and
phage 1358 as a model, we investigated the interaction
between the phage RBP and the pellicle hexasaccha-
ride of the host strain. A core trisaccharide (TriS),
derived from the pellicle hexasaccharide repeating
unit, was chemically synthesised, and the crystal
structure of the RBP/TriS complex was determined.
This provided unprecedented structural details of
RBP/receptor site-specific binding. The complete
hexasaccharide repeating unit was modelled and
found to aptly fit the extended binding site. The speci-
ficity observed in in vivo phage adhesion assays could
be interpreted in view of the reported structure. There-
fore, by combining synthetic carbohydrate chemistry,
X-ray crystallography and phage plaquing assays, we
suggest that phage adsorption results from distinct
recognition of the RBP towards the core TriS or the
remaining residues of the hexasacchride receptor.
This study provides a novel insight into the adsorption
process of phages targeting saccharides as their
receptors.
Introduction
The infection process of viruses is initiated by intermolecu-
lar interactions between the viral host recognition device
and a receptor usually located at the surface of the host
cell. This receptor can be a protein, a polysaccharide or
both. For example, using reversible attachment to cell wall
saccharides, bacterial viruses (bacteriophages or phages)
can scout the host cell surface to locate and irreversibly
bind to a specific receptor (Parent et al., 2014). Typical
examples include phage T5 (Plancon et al., 2002), which
infects Gram-negative Escherichia coli, and phage SPP1
(Alonso et al., 2006), which infects Gram-positive Bacillus

subtilis. Phage T5 uses the FhuA porin, an iron-importing
membrane protein, as a receptor, binding with sub-
nanomolar Kd (Breyton et al., 2013). Phage SPP1 uses the
protein YueB, a trans-membrane component of the type VII
secretion system, as a receptor and binds to it with sub-
nanomolar affinity (Sao-Jose et al., 2006).
On the other hand, most virulent phages infecting the
Gram-positive Lactococcus lactis utilise saccharides as
specific receptors (Chapot-Chartier et al., 2010; Bebeacua
et al., 2013; Ainsworth et al., 2014; Farenc et al., 2014;
Spinelli et al., 2014), including the predominant 936 group
and the rare 1358 group (Deveau et al., 2006). L. lactis is
the most important bacterial species used for cheese
manufacture, but the presence of ubiquitous virulent
phages in milk may lead to the lysis of lactococcal cells
thereby delaying the fermentation process. Accordingly,
hundreds of different lactococcal phages have been iso-
lated worldwide but surprisingly each replicate within a
specific set of L. lactis strains (Mahony and van Sinderen,
2012). The remarkable diversity of lactococcal phage host
ranges has been poorly studied but explanations are
beginning to emerge.
Lactococcal phages belong to the Caudovirales order
and, accordingly, these double-stranded DNA genome-
Accepted 19 February, 2015. *For correspondence. E-mail
cambillau
@afmb.univ-mrs.fr; [email protected]; Tel. 0033491825590;
Fax +35317162318. †These authors contributed equally to the
work.
Molecular Microbiology (2015) 96(4), 875–886 ■
doi:10.1111/mmi.12978
First published online 16 March 2015

© 2015 John Wiley & Sons Ltd
mailto:[email protected]
containing bacterial viruses use the receptor-binding pro-
teins (RBP) located at tip of their tail to recognise receptors
on the host (Spinelli et al., 2014). The structure of several
RBPs from various lactococcal phages has been solved
(Ricagno et al., 2006; Spinelli et al., 2006a,b; Farenc et al.,
2014). Among others, it was found that lactococcal RBPs
are highly diversified; however, RBPs share a modular
structure and a saccharide-binding site located at the
C-terminal end that is critical for host binding. This assort-
ment of phage RBPs is likely related to the diversity of the
phage receptors at the surface of L. lactis cells.
The exact nature of these cell receptors, however, has
long been a matter of debate. It was proposed that they
may be phosphosaccharides, such as cell wall lipotei-
choic acids (Tremblay et al., 2006). These polymers have
been found to serve as receptors for various Staphylococ-
cus aureus phages (Xia et al., 2010; 2011) but are not
diverse enough structurally to promote finely tuned host
specificity.
In 2010, a new phosphosaccharide pellicle enveloping
L. lactis cells was discovered, and its structure was
determined for strain MG1363 (Andre et al., 2010;
Chapot-Chartier et al., 2010). The MG1363 pellicle is
made of a repeat of a phosphohexasaccharide, possibly
linked covalently to the cell wall. Very recently, two other
pellicles were analysed, those of L. lactis strain 3107

(Ainsworth et al., 2014) and strain SMQ-388 (Farenc et al.,
2014). The differences in their structures (Fig. 1) were
ascribed as the trigger of the fine specificity of lactococcal
phages for their host (Ainsworth et al., 2014). Comparative
analyses led to the hypothesis that a trisaccharide motif
(GlcNAc-Galf-GlcNAc-1P or GlcNAc-Galf-Glc-1P) within
the pellicle is the core of these phage receptors, whereas
strain specificity may be defined by the remaining compo-
nents of the pellicle hexasaccharide (Farenc et al., 2014)
(All the saccharide residues in this report are pyranosides,
with the exception of the furanoside Galf).
We reported recently the X-ray structures of phage
1358 RBP in complex with glycerol, GlcNAcp and Glcp-1P
(Farenc et al., 2014). These structures identified two sites
at the RBP surface, ∼ 8 Å apart, one accommodating a
GlcNAc monosaccharide, the other a GlcNAc or a Glc-1P
monosaccharide. Given that GlcNAc and Glc-1P are com-
ponents of the polysaccharide pellicle of L. lactis SMQ-
388 (the host of phage 1358), a Galf sugar bridging the
two GlcNAc was modelled, reasoning that the trisaccha-
ride motif GlcNAc-Galf-GlcNAc-1P (or Glc-1P) may be
common to receptors of genetically distinct lactococcal
phages p2, TP901-1 and 1358. Here, we experimentally
support this hypothesis by first synthesising the con-
served core trisaccharide, and then, demonstrating that it
binds to the phage RBP at the same locations as the
monosaccharide complexes and in a mode comparable,
but slightly distinct, to the previous in silico model. We
also propose a molecular model of the complete hexasac-
charide binding.
Results
Chemical synthesis of a conserved core trisaccharide

A trisaccharide commonly expressed in the polysaccha-
ride pellicle repeating units of L. lactis strains (Fig. 1,
bottom) was chemically synthesised from three monosac-
charides, D-GlcNAc, D-Galf, and D-Glc, each possessing
varied protecting group patterns. As the glucose moiety is
α-linked in the phosphodiester bridges, the trisaccharide
was synthesised as its α-methyl glucoside in order to
maintain the natural stereochemical configuration at the
reducing end.
Following a previously reported strategy (Sato et al.,
2003), the galactofuranosyl intermediate 4 was syn-
thesised from commercially available 1,2:5,6-di-O-
isopropylidene-α-D-glucofuranose (1) (Fig. 2A). Com-
pound 1 was first treated with triflic anhydride to form
the 3-O-triflate derivative 2, which was subsequently
heated in DBU/DMSO to give the elimination compound 3
with a 91% yield. The 1,2:5,6-di-O-isopropylidene-α-D-
galactofuranose (4) was then afforded following the regio-
and stereoselective hydroboration-oxidation of 3. The
Fig. 1. Structure of the phosphosaccharide
repeating motifs of the pellicle of the L. lactis
strains MG1363 (Chapot-Chartier et al.,
2010), 3107 (Ainsworth et al., 2014) and
SMQ-388 (Farenc et al., 2014) (from top to
bottom) and of the TriS molecule. The three
saccharides forming the core of the pellicle
are boxed.
876 O. McCabe et al. ■
© 2015 John Wiley & Sons Ltd, Molecular Microbiology, 96,
875–886

5,6-O-isopropylidene acetal was selectively cleaved
before treatment with NaH and benzyl bromide in DMF to
obtain compound 6. Hydrolysis of the 1,2-isopropylidene
acetal in 6, followed by benzoylation with BzCl in pyridine,
gave compound 7 in an 89% yield. The corresponding
thioglycoside was formed by treating 7 with ethanethiol and
BF3·OEt2, yielding ethyl 2-O-benzoyl-3,5,6-tri-O-benzyl-1-
thio-D-galactofuranoside (8) (Fig. 2B) as an anomeric
mixture (α:β, 1:5). The highly reactive nature of compound
7 often resulted in di-thiolation at the anomeric carbon
during thioglycoside formation.
The 2-O-benzoyl ester in compound 8 has two functions:
to control by means of neighbouring group participation the
stereochemistry of newly formed glycosidic bonds in gly-
cosylation using 8 as a glycosyl donor, and to allow for
selective deprotection in the presence of benzyl ethers to
form a 2-OH glycosyl acceptor (Fig. 2B). The NIS/AgOTf-
promoted glycosylation in CH2Cl2 (Konradsson et al.,
1990) of methyl glucoside 9 (Zissis and Fletcher, 1970;
Garegg et al., 1990) with donor 8 gave the β-linked disac-
charide 10 with a 92% yield. Disaccharide acceptor 11
was acquired following debenzoylation using standard
Zemplén conditions. Acceptor 11 was then coupled with
donor 12 (Cirla et al., 2004) in a subsequent NIS/AgOTf-
promoted glycosylation affording the fully protected trisac-
charide 13 with an 82% yield.
The phthalimido group was removed using ethylenedi-
amine in ethanol at an elevated temperature (→14) before
Fig. 2. Chemical synthesis of a conserved
core trisaccharide.

A. (i) Tf2O, dry pyridine, dry CH2Cl2, −20°C,
1 h; (ii) DBU, dry DMSO, 85°C, 1.5 h; (iii) 1.
BH3·THF, dry THF, 40°C, 4 h, 2. H2O, NaOH,
H2O2, rt, 30 min; (iv) 70% AcOH, rt, 6 h; (v)
BnBr, NaH dry DMF, rt, 24 h; (vi) (a) 85%
AcOH, 70°C, 24 h, (b) BzCl, dry pyridine, rt,
24 h; (vii) EtSH, BF3·OEt2, dry CH2Cl2, 0°C,
15 min.
B. (i) NIS, AgOTf, 4Å MS, dry CH2Cl2, 0°C,
45 min; (ii) NaOMe, dry MeOH, rt, 24 h; (iii)
NIS, AgOTf, 4Å MS, dry CH2Cl2, −15°C,
40 min.
C. (i) (a) EDA, EtOH, 70°C, 24 h, (b) Ac2O,
pyridine, rt, 3 h; (ii) Pd/C, H2 (20 bar),
EtOAc/EtOH/H2O (6:4:1), rt, 48 h. The
subsequent steps of the synthesis are
indicated by letters (i), (ii) and (iii).
Receptor binding to lactococcal phage 1358 877
875–886
N-acetylation was carried out to produce trisaccharide 15
in an 83% yield over the two steps (Fig. 2C). Complete
deprotection was achieved via hydrogenolysis of the
benzyl ethers using Pd/C under a hydrogen atmosphere
(20 bar). This afforded the desired trisaccharide methyl
(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-(β-D-
galactofuranosyl)-(1→6)-α-D-glucopyranoside (16, TriS)
in a 95% yield.
Taken altogether, a trisaccharide (TriS) commonly found
in the polysaccharide pellicle repeating units of L. lactis

strains was chemically synthesised as its α-methyl glyco-
side in six steps from the three monosaccharide interme-
diates 8, 9 and 12 in an overall yield of 51%.
Trisaccharide binding to the phage 1358 RBP
As with other lactococcal RBPs, the RBP of phage 1358
assembles as a trimer, with each monomer having a
modular structure such as an embedded N-terminal
domain and a C-terminal receptor binding ‘head’ domain
(Farenc et al., 2014). The crystal structure of phage 1358
RBP in complex with TriS was determined by X-ray diffrac-
tion at 2.10 Å resolution. Two RBP monomers are present
in the asymmetric unit, each generating a biologically
relevant trimer by applying crystallographic threefold sym-
metry. A clear electron density was identified in the head
domain of both independent monomers in the asymmetric
unit, which was large enough to fit the length of approxi-
mately three sugar moieties. The molecular structure of
TriS obtained from CCP4 Sketcher module (Winn et al.,
2011) was readily fitted in the electron density map without
ambiguity for the direction of the saccharide chain. Refine-
ment and manual rebuilding (Table 1) yielded an excellent
model of TriS, although the C5, O5, C6 and O6 atoms of
Galf were poorly defined in the electron density map
(Fig. S1) and had higher B-factors than the rest of TriS. TriS
is located in the middle of the RBP head domain, in a deep
crevice (Fig. 3A) in which monosaccharides in complex
with the phage RBP were observed (Farenc et al., 2014).
Worth noting, TriS occupies about half of this crevice.
Furthermore, the cavity becomes wider to the left of the
GlcNAc 3 moiety (on Fig. 3B). The role of the remaining
free volume of the cavity is discussed later in the text.
The TriS binds to the phage RBP through numerous
interactions and its buried surface area in the complex

is 254 Å2, 55% of its total accessible surface. Although
the Glc 1 and GlcNAc 3 moieties strongly interact with
the amino acids of the binding site, the middle Galf is
mostly exposed and does not display any direct con-
tact with the RBP (Fig. 4, S2). As often observed in
other polysaccharide–protein interactions (Bourne et al.,
1994a,b), TriS is wrapped around two hydrophobic resi-
dues, Phe 240 and 243 (Table 2). Several TriS–RBP
interactions are also mediated by chains of water mol-
ecules, also a common feature of protein–sugar interac-
tions (Bourne et al., 1990). Glc 1 is strongly anchored in
the binding site by hydrogen bonds between O1 and Ser
291 OH, O2 and the guanidinium group of Arg 23, and O3
and the NH2 group of Gln 341. TriS O4 interacts with a
water molecule, itself strongly anchored by Arg 312 and
Asn 289. The N-acetyl group of GlcNAc 3 is hydrogen-
bound to the NH2 group of Gln 345 and the O2 atom with
the main-chain NH of Ser 202. The O3 atom interacts
with a water molecule bound to the carbonyl group of Val
200. Two water molecules are bound to the O4 and O5
atoms of Galf that are further hydrogen-bound to other
atoms of TriS, Glc O5 and GlcNAc O7, respectively, con-
tributing to the stabilisation of the saccharide geometry.
When comparing the position of TriS residues 1 and 3
to the previously determined positions of the monosac-
charides bound to RBP (Farenc et al., 2014), we noticed
that the GlcNAc 3 occupies exactly the same position
and has the same orientation as the second GlcNAc from
the GlcNAc complex (PDB 4L92) (Fig. 5A). In contrast,
the TriS Glc 1 residue is rotated clockwise by ∼ 60°
around the sugar center, as compared with the three free
saccharides found at this position, GlcNAc, Glc-1P and
Table 1. Data collection and refinement statistics of the phage

1358
RBP crystals.
Data collection ORF20-TriS
PDB 4RGA
Source Soleil PX 1
Space group, cell a = b = c (Å) P213, 166.4
Resolution limitsa (Å) 48.0–2.10
(2.16–2.10)
Rmeasa (%) 11.0 (75.0)
CC(1/2) 99.8 (85)
Total reflectionsa 1012656 (82404)
Unique reflectionsa 89232 (7218)
Mean((I)/sd(I))a 12 (2.1)
Completenessa (%) 99.9 (98.5)
Multiplicitya 11.4 (11.4)
Refinement
Resolutiona (Å) 48.0–2.10
(2.15–2.10)
Nr of reflectionsa 89232 (6540)
Nr protein/water/ligand 6078/927/76
Nr test set reflections 4462 (327)
Rwork/Rfreea (%) 18.1/19.2
(18.9/20.6)
r.m.s.d.bonds (Å)/angles (°) 0.010/1.12
B-wilson Å2 39.7
B-atoms (A/B) 46.0/45.5
B-ligands (A/B) 61.0/58.3
B-Waters 58.0
Ramachandran: preferred/allowed/outliers b (%) 97.0/2.48/0.52

a. Numbers in brackets refer to the highest resolution bin.
b. Amino-acids Tyr182 and Gly317 are slightly outside the
allowed
area of the Ramachandran plot but have a clear electron density
map.
In complex with the trisaccharide βGlcNAc-Galf-αGlcNAc-
OMe.
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GlcNAc-1P [PDB IDs 4L92, 4L97, 4RGG (Farenc et al.,
2014)]. Therefore, each Ci atom of the TriS Glc 1 residue
occupies the position of the Ci+1 atom in the monosaccha-
ride complexes. This rotation is indeed induced by the
geometry constraint arising from the Glc-Galf linkage. It is
worth noting that, despite this rotation, both saccharides
keep the same hydrogen-bonds with the protein, but not
with the same OH groups. Furthermore, the in silico sub-
stitution of a NAc moiety at C2 of TriS Glc 1, presenting a
GlcNAc residue in position 1, did not provoke steric
clashes with the protein, aptly positioning the NAc group
in a small pocket (Fig. 5B). The O7 atom of NAc replaces
a water molecule in the experimental structure and pro-
vides an extra hydrogen-bond with Asn 289 NH2 group.
Noteworthy, superposition of the RBP structure in
complex with TriS with those of the complexes with
GlcNAc, Glc1P and GlcNAc-1P yielded very low root
mean square (r.m.s.) deviations, between 0.09 to 0.15 Å.

A closer look at the saccharide binding sites confirm their
lack of conformational changes, also including the side-
chains of the binding site. The r.m.s. deviation observed
with the native RBP (in another crystal form) is slightly
larger (0.45 Å). However, the saccharide binding site resi-
dues are superimposable, including their side-chains.
Hexasaccharide modeling and docking into 1358
RBP binding site
We mentioned above that the TriS seemed to occupy only
a part of the binding crevice volume identified at the RBP
surface (Figs 3 and 6A). Because the structure of the
surface pellicle of L. lactis SMQ-388 (Fig. 1, top), the host
of phage 1358, has been determined (Farenc et al., 2014),
we constructed this hexasaccharide in silico by adding
monosaccharides 4, 5 and 6 (Fig. 6B) to TriS with the
correct glycosidic linkages aided by the CPP4 option
‘sketcher’ (Winn et al., 2011) and with Coot (Emsley et al.,
2010). The orientation of the 6-OH of GlcNAc 3 was turned,
as this group, turned inside the crevice, had to be substi-
tuted with Glc 6 (Figs 3B and 6A). The SMQ-388 hexasac-
charide was the docked into the RBP crevice, and
REFMAC5 (Murshudov et al., 2011) was used to optimise
the docking by removing close contacts. The resulting
model showed that the overall position of TriS remained
unchanged and that the three added saccharides comple-
mented the binding cavity shape. At the reducing extremity,
the OMe moiety, a PO3 group in the pellicle, is favorably
exposed to solvent, allowing the next pellicle phos-
phohexasaccharides to be positioned freely (Fig. 6B, red
arrow right). At the non-reducing end, the O3 atom of
GlcNAc 5 is also accessible to solvent, indicating that the
pellicle chain can be continued (Fig. 6B, red arrow left).
Phages–host interactions

Three different surface pellicle polysaccharides have
been identified and analysed in three lactococcal strains,
Fig. 3. X-ray structure of the phage 1358
RBP in complex with the TriS molecule.
Representation of the TriS molecule (sticks) in
the complete trimeric RBP surface (blue,
green and pink). The head domain is situated
above the horizontal blue line. Inset: close-up
of the binding site. GlcNAc, Galf and Glc
saccharide atoms are identified by their
number. Figure made with Pymol (Pymol,
2014).
This figure is available in colour online at
wileyonlinelibrary.com.
Table 2. Interactions between bound saccharides and the phage
1358 RBP.
GlcNAc-Galf-
Glc-OMe BSA
Å2; % of ASA
GlcNAc hydrogen
bonds length (Å)
Total 254; 55%
Val 200 # 13.4
Gln 201 # 4.4
Ser 202 # 5.4 N-H-OH23;2.8
Arg 237 • 12.6; 24% NH1,2-HO3 3.1; 3.2
Phe 240 • # 37; 50% C=O-OH24; 2.9. C=O-OH28; 2.8
Asp 243 #
Asn 244 • # 3.4; 75% C=O-HO3; 3.35
Asn 289 • 25; 41%

Ser 291 • 22; 25% Oγ-OH1; 2.81
Ala 292 • 13; 52%
Asn 341 • 8.7; 95% C=O-HO3; 2.6
Phe 343 • 16; 78%
Gln 345 # 11; C=O-HO27; 2.8
ASA, accessible surface area; BSA, buried surface area.
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http://wileyonlinelibrary.com
Fig. 4. Details of the molecular interaction of
TriS with the active site side-chains.
A. Ligplot+ drawing (Laskowski and Swindells,
2011) of the interactions of Tris with protein
and water. Only the ligand and protein bound
water molecule are displayed. They are
conserved between the two independent
sites.
B. The TriS moiety is represented as sticks
(C, yellow; O, red; N, blue). The RBP
backbone is drawn in cartoon representation
(green), the side chains of the RBP binding
sites residues are represented as sticks (C,
white; O, red; N, blue). Blue transparent lines
illustrate the hydrogen bonds. The red
spheres are water molecules. Their lengths
are indicated in Å. Figure made with Pymol
(Pymol, 2014).

875–886
MG1363, 3107 and SMQ-388. As indicated in Fig. 1, the
pellicle of strains L. lactis 3107 and SMQ-388 share almost
the same trisaccharide core (GlcNAc-Galf-GlcNAc1P),
whereas strain MG1363 is slightly different (GlcNAc-Galf-
Glc1P). Phages 1358 infect its host SMQ-388 as well as
strain 3107 but not MG1363 (data not shown). On the other
hand, phage p2 infects only the strain MG1363 and phage
TP901-1 only the strain 3107(data not shown).
We then performed phage adsorption assays in which
we measured the percentage of phage adsorbed to the
cells after an incubation of 15 min (see Materials and
methods). As expected, phage p2 adsorbed only to its host
strain MG1363, whereas TP901 adsorbed mainly to its
host 3107 (Table 3). Phage 1358 had a lower adsorption to
its hosts SMQ-388 as compared with the other two phage–
host pair. In agreement, the adsorption of phage 1358 to
L. lactis 3107 was also low (Table 3). Of note the adsorp-
tion level increased when phage 1358 was incubated for a
longer period of time with its host (data not shown).
Discussion
Phage–host interactions are influenced by a number of
factors including the presence of various phage defense
mechanisms in lactococcal strains, which may prevent the

lysis of a phage-infected cell (Labrie et al., 2010; Samson
and Moineau, 2013). Recognition and binding of the phage
to its host cell is key to the initiation of these interactions.
TriS saccharide synthesis
Although the hexa/pentasaccharide motifs of the three
pellicles could be purified directly from the L. lactis strains,
the quantities available were not enough for X-ray crys-
tallographic studies. Thus, we decided to embark on a
synthetic approach, which comprise the ability to generate
not only the pellicle motifs, but also fragments and deriva-
tives thereof. The TriS synthesis, reported here, shows
the usefulness of this approach in an efficient synthesis.
Starting from appropriately protected monosaccharide
building blocks, the target trisaccharide was produced on
a ∼ 20 mg scale. These building blocks, with minor adjust-
ments, are now being further used in the continued syn-
theses of other pellicle oligosaccharide structures to
assist in future structural and biophysical studies.
The RBP structure of phage 1358 and its
saccharide-binding site
The RBP from phage 1358 displays large differences from
those of phages p2 and TP901-1 (Fig. S3A–C). In particu-
lar, the receptor binding site has been located between two
monomers in phages p2 and TP901-1 (Fig. S3D and E)
while it is in the middle of the monomers of the RBP of
phage 1358 (Fig. S3F). The saccharide-binding site of
phage 1358 is an elongated crevice, which can be filled in
part by our synthetic TriS. This TriS defines a core trisac-
charide present in the three wall polysaccharide (WPS)
identified to date, the position 1 being Glc-1P or GlcNAc-
Fig. 5. Analysis of the TriS geometry.

A. TriS geometry and position compared to those of GlcNAc
monosaccharides bound to the RBP of 1358. Sticks
representations and atom mode coloring (Tris: C, white; O, red;
N,
blue. GlcNAc: C, orange; O, red; N, blue ).
B. TriS is compared with a model in which Glc 1 has been
replaced
by a GlcNAc saccharide. The RBP surface is green. Stick
representations and atom mode coloring (TriS: C, white; O, red;
N,
blue. GlcNAc Model: C, yellow; O, red; N, blue). Figure made
with
Pymol (Pymol, 2014).
Table 3. Adsorption assay of phages p2, TP901-1 and 1358 on
the
Lactococcus lactis strains MG1363, 3107 and SMQ-388.
Percentage of phage adsorption
Strain/phage p2 TP-901 1358
MG1363 82.7 ± 9.4 21.5 ± 5.0 18.7 ± 8.0
3107 0 85.2 ± 7.8 31.5 ± 11.2
SMQ-388 0 5.8 ± 5.0 60.4 ± 9.8
Bold numbers refer to the host’s specific phage.
875–886

1P. TriS (or TriS-NAc) would therefore be a seeding
module, attaching to all RBPs binding site, whereas selec-
tivity might occur from the rest of the hexasaccharide
pellicle motif. In this context, we notice that two main
differences between the three WPS: position 4 exhibits a
Rha (MG1363) or a Galf (SMQ-388, 3107), both 1→3
linked to the core trisaccharide with elongation through
position 3. However, with the Galf residue being a
5-membered ring, the angle formed by the saccharides
bound to Galf 4 is smaller than in the case of a pyranose
saccharide (6-membered ring). The Galf at position 4 may,
therefore, introduce steric clashes with the RBP binding
site of a WPS possessing GlcNAcp at this position. The
second large difference is the substitution of a Glc at 6-OH
of GlcNAc 3. This substitution would introduce steric
clashes in a site for which the specific WPS is not substi-
tuted. In contrast, binding a non-substituted WPS to a site
accepting Glc substituted WPS would be less dramatic, the
only difference being probably a small loss in extra binding
energy arising from interactions of the branched Glc in the
RBP site.
The adsorption of phage 1358 to L. lactis strains
The adsorption of phage 1358 at the surface of the L. lactis
strain SMQ-388 results from a compatibility between the
pellicle repeating phosphohexasaccharide and the binding
crevice of the phage RBP. The hexasaccharide spans the
width of the binding crevice, leaving the terminal ends
exposed and allowing the rest of the polymer to be posi-
tioned freely (Fig. 6B). Furthermore, all the nooks of the
binding cavity are filled, leading to optimal interaction
energy. Concerning the lower adsorption of phage 1358 at
the surface of its other host L. lactis strain 3107, we noticed

that the only differences between both receptors are the
absence of a branched Glc 6 and a Glc 5 instead of GlcNAc
5 in strain 3107. We suggested above that the absence of
branched Glc should have minor effects (less interaction
energy). Furthermore, in our hexasaccharide model, a NAc
group at position 2 of Glc 5 would be exposed to solvent.
Phage 1358 does not infect L. lactis MG1363, and its
adsorption level was the lowest (Table 1). We noticed
a unique difference between both saccharide motifs
(besides Glc 1), a Rha instead of Galf in strain MG1363.
The angle between the saccharides attached to Rha
should be larger than for Galf, hence we observe in our
model that Rha 4 and GlcNAc 5 of the MG1363 pellicle are
further from the RBP crevice and thus less prone to estab-
lishing favourable interactions. However, this kind of sub-
stitution (not the contrasting one) does not induce steric
clashes but only a milder loss of favourable interactions. As
we do not know the structure of a complex between TriS
Fig. 6. A model of the SMQ-388
hexasaccharide in interaction with the RBP.
A. The original TriS structure in sphere
representation at the RBP surface. Inset:
close-up of the binding crevice; saccharides
are numbered 1–3 and their sequence is
given below.
B. The modelled SMQ-388 hexasaccharide
structure in sphere representation at the RBP
surface. Inset: close-up of the binding crevice;
saccharides are numbered 1–6 and the
sequence of the modelled motif is given
below. The two red arrows indicate the spread
of the polysaccharide at the reducing and
non-reducing end. Figure made with Pymol
(Pymol, 2014).

875–886
and the p2 and TP901-1 RBPs, analysis of the adsorption
data in terms of pellicle/RBP interaction is not possible at
this time.
Conclusion
In summary, these results provide a better understanding
of the adhesion properties of phage 1358 towards its
L. lactis host strains, as well as phage–host interactions in
general. Overall, our data suggest that the binding
mechanism of a lactococcal phage to its host may involve
two different interactions of the RBP with the hexasaccha-
ride receptor: the TriS moiety, being similar in the three
receptors sequenced, may provide a non-specific initial
interaction, whereas the rest of the hexasaccharide may
increase the interaction for strain-specific phages, or
abolish it for non-specific phages.
These results also provide a stimulating illustration of
what can be achieved by combining synthetic carbohy-
drate chemistry, X-ray protein crystallography analysis and
in vivo viral infections. Finally, the TriS synthesis paves the
way for the synthesis of larger pellicle saccharides with
different specificities, making it possible to experimentally
verify our suggested recognition mechanism.
Experimental procedures
Chemical synthesis of a conserved core trisaccharide

Unless noted, chemical reagents and solvents were used
without further purification from commercial sources. Reac-
tions were magnetically stirred. Concentration in vacuo was
generally performed using a Buchi rotary evaporator. The
1H/13C NMR spectra (δ in ppm, relative to TMS in CDCl3)
were
recorded with Varian spectrometers (Varian, Palo Alto, CA,
USA) (400/101 MHz or 500/125 MHz) at 25°C. Assignments
were aided by 1H-1H and 1H-13C correlation experiments.
HRMS spectra were recorded on a micromass LCT instru-
ment from Waters. Optical rotations were recorded on a
Perkin-Elmer polarimeter (Model 343) at the sodium D-line
(589 nm) at 20°C using a 1 dm cell and are not corrected.
Silica gel chromatography was carried out using Davisil
LC60A (Grace tech., Columbia, MD, USA) SiO2 (40–63 μm)
silica gel. All reactions were monitored by thin-layer chroma-
tography (TLC). TLC was performed on Merck DC-Alufolien
plates precoated with silica gel 60 F254. They were visual-
ised with UV-light (254 nm) fluorescence quenching, and/or
by charring with an 8% H2SO4 dip (stock solution: 8 ml conc.
H2SO4, 92 ml EtOH), and/or ninhydrin dip (stock solution:
0.3 g ninhydrin, 3 ml AcOH, 100 ml EtOH).
1,2:5,6-Di-O-isopropylidene-3-O-triflate-α-D-glucofuranose
(2). Pyridine (1.4 ml, 17.7 mmol) was added to a solution
of 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose 1 (2.0 g,
7.7 mmol) in dry CH2Cl2 (60 ml). The mixture was cooled to
−20°C, and a solution of triflic anhydride (1.6 ml, 9.2 mmol) in
CH2Cl2 (20 ml) was added over 30 min. The reaction was
stirred at −20°C for an additional 30 min, before being diluted
with CH2Cl2 (60 ml) and washed successively with H2O
(60 ml), sat. aq. NaHCO3 (60 ml) and H2O (60 ml). The
organic layer was dried over MgSO4, filtered and concen-
trated under diminished pressure to give a yellow solid. The

crude product was used for the next step without purification.
Rf 0.79 (toluene/EtOAc, 1:1 +1% Et3N).
3-Deoxy-1,2:5,6-di-O-isopropylidene-α-D-erythro-hex-3-eno-
furanose (3). Compound 2 (3.0 g, 7.7 mmol) was dissolved
in dry DMSO (90 ml). DBU (1.4 ml, 9.2 mmol) was added,
and the reaction was stirred at 85°C for 1.5 h. Upon com-
pletion, the mixture was poured onto H2O (200 ml) and
extracted with EtOAc (6 × 50 ml). The organic phases were
combined, dried over MgSO4, filtered and evaporated in
vacuo. The crude product was then purified by silica gel
chromatography (toluene/EtOAc, 6:1 v/v + 1% Et3N), which
gave compound 3 (1.69 g, 91% over two steps) as an off-
white solid.
1,2:5,6-Di-O-isopropylidene-α-D-galactofuranose (4). Com-
pound 3 (200 mg, 0.8 mmol) was dissolved in dry THF
(20 ml) and cooled to 0°C. A solution of BH3·THF in THF (1M,
1.7 ml) was added, and the resulting solution was stirred at
40°C. After stirring for 3 h, a substantial amount of starting
material was still detected by TLC (toluene/EtOAc, 4:1 v/v).
The reaction was cooled to 0°C and additional BH3·THF in
THF (1M, 2.0 ml) was added. The reaction was stirred at
40°C for 1 h. Once complete, the solution was cooled to 0°C
and H2O (0.8 ml), 10% aq NaOH (2.4 ml) and 35% aq H2O2
(6.0 ml) were sequentially added. The mixture was stirred at
room temperature for 30 min. H2O (20 ml) was added, and
the aqueous solution was extracted with CH2Cl2 (3 × 20 ml).
The organic phase was combined, dried over MgSO4, filtered
and concentrated in vacuo. Purification by silica gel chroma-
tography (toluene/EtOAc, 2:1 v/v) was carried out, yielding 4
(131 mg, 61%) as a white solid.
1,2-O-Isopropylidene-α-D-galactofuranose (5). Compound 4
(2.0 g, 7.7 mmol) was treated with 70% aq AcOH (30 ml). The
solution was stirred at room temperature for 6 h. Once com-

plete, the mixture was concentrated in vacuo followed
by co-evaporation with H2O and toluene, successively. The
crude product was then purified by silica gel chromatography
(EtOAc/MeOH, 9:1 v/v) giving compound 5 (1.33 g, 79%) as
a white solid.
1,2-O-Isopropylidene-3,5,6-tri-O-benzyl-α-D-galactofuranose
(6). A solution of compound 5 (667 mg, 3.0 mmol) and
BnBr (2.2 ml, 18.2 mmol) in dry DMF (20 ml) was
added to a cooled suspension of 60% NaH (848 mg,
21.0 mmol) in dry DMF (10 ml) at 0°C. The reaction was
stirred at room temperature overnight. MeOH was added at
0°C, to quench excess NaH, and the mixture was concen-
trated in vacuo. The residue was diluted with H2O (40 ml)
and extracted with CH2Cl2 (10 ml × 4). The organic phases
were combined and washed with sat. aq. NaHCO3
(2 × 20 ml) and H2O (20 ml). The organic layer was dried
over MgSO4, filtered and concentrated in vacuo. Purification
by silica gel chromatography (toluene/EtOAc, 15:1 v/v)
afforded 6 (1.38 g, 93%).
875–886
1,2-O-Benzoyl-3,5,6-tri-O-benzyl-D-galactofuranose
(7). Compound 6 (1.83 g, 3.7 mmol) was dissolved in 85%
aq AcOH (24 ml) and stirred at 70°C overnight. The solution
was poured onto excess sat. aq NaHCO3 and extracted with
CH2Cl2 (20 ml × 6). The organic extracts were dried over
MgSO4, filtered and concentrated in vacuo. The crude
product was used for the next step without purification. Rf
0.38 (toluene/EtOAc, 6:1). BzCl (1.7 ml, 15.0 mmol) was

added to a cooled solution (0°C) of the crude product in dry
pyridine (20 ml). The resulting mixture was stirred at room
temperature overnight. The solvent was evaporated under
diminished pressure and the residue was re-dissolved in
EtOAc (40 ml). The solution was washed with H2O (20 ml),
1M HCl (2 × 20 ml), sat aq NaHCO3 (2 × 20 ml) and H2O
(20 ml). The organic phase was then dried over MgSO4,
filtered and concentrated in vacuo. Purification by silica gel
chromatography (cyclohexane/EtOAc, 12:1–10:1 v/v) gave 7
(2.23 g, 91%) as a white solid.
Ethyl 2-O-benzoyl-3,5,6-tri-O-benzyl-1-thio-D-galactofurano-
side (8). Compound 7 (2.0 g, 3.0 mmol) was dissolved in
dry CH2Cl2 (30 ml) and cooled to 0°C. Ethanethiol (1.1 ml,
15.2 mmol) was added, and the mixture was stirred for
15 min. BF3·OEt2 (0.6 ml, 4.6 mmol) was added dropwise,
and the reaction was stirred for 1 h at 0°C. Upon completion,
the solution was neutralised with Et3N, and the solvent
was concentrated in vacuo. Silica gel chromatography
(cyclohexane/EtOAc, 20:1–15:1 v/v) of the crude product
gave 8 (1.14 g, 63%) as an anomeric mixture (β:α = 5:1).
Methyl (2-O-benzoyl-3,5,6-tri-O-benzyl-β-D-galactofuranosyl)-
(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (10). A solu-
tion of methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside
(9,87 mg, 0.19 mmol), compound 8 (135 mg, 0.23 mmol)
and 4Å MS (350 mg) in dry CH2Cl2 (5 ml) was cooled to 0°C.
The mixture was stirred for 15 min before addition of
N-iodosuccinimide (63 mg, 0.28 mmol) and AgOTf (12 mg,
0.05 mmol). The reaction was stirred at 0°C for 45 min and
then neutralised with Et3N. The mixture was filtered through
Celite (Imerys Mineral, CA, USA) and washed with 10% aq
Na2S2O3 (5 ml) and H2O (5 ml). The organic phase was dried
over MgSO4, filtered and concentrated in vacuo. The crude
product was subjected to silica gel chromatography
(cyclohexane/EtOAc, 10:1–5:1 v/v) to obtain disaccharide 10

(173 mg, 92%).
Methyl (3,5,6-tri-O-benzyl-β-D-galactofuranosyl)-(1→6)-2,3,
4-tri-O-benzyl-α-D-glucopyranoside (11). Disaccharide 10
(158 mg, 0.16 mmol) was dissolved in dry MeOH (1.6 ml) and
cooled to 0°C. NaOMe was added until a pH of 12 was
reached. The solution was stirred at room temperature over-
night. Dowex-50WX8 resin was used to neutralise the reac-
tion. The mixture was filtered and concentrated in vacuo.
Purification by silica gel chromatography (toluene/EtOAc,
8:1–6:1 v/v) gave 11 (122 mg, 86%).
Methyl (3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-β-D-glu-
copyranosyl)-(1→2)-(3,5,6-tri-O-benzyl-β-D-galactofurano-
syl)-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (13). A
solution of ethyl 3,4,6-tri-O-benzyl-2-deoxy-2-phthtalimido-1-
thio-β-D-glucopyranosyl 12 (88 mg, 0.14 mmol), disaccha-
ride acceptor 11 (85 mg, 0.10 mmol) and 4Å MS (230 mg) in
dry CH2Cl2 (4.5 ml) was cooled to −15°C. The mixture was
stirred for 15 min before addition of NIS (32 mg, 0.14 mmol)
and AgOTf (6 mg, 0.02 mmol). The reaction was stirred
at −15°C for 40 min and then neutralised with Et3N. The
mixture was filtered through Celite and washed with 10% aq
Na2S2O3 (3 ml) and H2O (3 ml). The organic phase was dried
over MgSO4, filtered and concentrated in vacuo. The crude
product was subjected to silica gel chromatography
(cyclohexane/EtOAc, 8:1–6:1–5:1 v/v) to obtain trisaccharide
13 (112 mg, 82%).
Methyl (2-amino-3,4,6-tri-O-benzyl-2-deoxy-β-D-glucopy-
ranosyl)-(1→2)-(3,5,6-tri-O-benzyl-β-D-galactofuranosyl)-
(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (14). Ethyl-
enediamine (0.14 ml, 2.1 mmol) was added to a solution of
disaccharide 13 (76 mg, 0.05 mmol) in EtOH (4 ml). The
reaction was stirred at 70°C overnight. The mixture was

co-evaporated with acetonitrile under diminished pressure.
Purification by silica gel chromatography (toluene/EtOAc, 6:1
v/v +1% Et3N) afforded 14 (57 mg, 83%).
Methyl (2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-glu-
copyranosyl)-(1→2)-(3,5,6-tri-O-benzyl-β-D-galactofurano-
syl)-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (15).
Acetic anhydride (8 μl, 0.08 mmol) was added to a solution of
trisaccharide 14 (54 mg, 0.04 mmol) in pyridine (0.5 ml) at
room temperature. After 3 h of stirring, the solvent was
removed under reduced pressure, and the residue was
re-dissolved in CH2Cl2 (2 ml). The solution was washed with
1M HCl (1 ml), sat aq NaHCO3 (1 ml) and H2O (1 ml). The
organic phase was dried over MgSO4, filtered and concen-
trated in vacuo. Silica gel chromatography (toluene/EtOAc,
6:1 v/v) of the crude product gave compound 15 (51 mg,
91%).
Methyl (2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-
(β-D-galactofuranosyl)-(1→6)-α-D-glucopyranoside (16).
Pd/C 10% molar (33 mg, 0.31 mmol) was added to a solution
of trisaccharide 15 (47 mg, 0.034 mmol) in EtOAc/EtOH/H2O
(0.5 ml, 6:4:1). The reaction was stirred vigorously under an
atmosphere of H2 gas (20 bar) at room temperature for 2
days. The mixture was filtered through a pad of Celite and
concentrated in vacuo. Reverse phase chromatography gave
trisaccharide 16 (19 mg, 95%) as a white solid.
RBP/Trisaccharide crystallisation. The orf-20 of phage 1358
was cloned into the Gateway™ (Invitrogen, Grand Island, NY,
USA) destination vector pETG-20A for protein production in
E. coli BL21, purified by Ni affinity, and gel filtration chroma-
tography according to standard procedures (Vincentelli et al.,
2003; 2005) as previously described (Farenc et al., 2014). A
solution of RBP in 10 mM HEPES, pH 7.5, 150 mM NaCl was
mixed with TriS (dissolved in water) to final concentrations of

13 mg ml−1 of RBP and 100 mM of TriS. Cocrystals were
obtained by mixing 300 nl of the protein/TriS solution with a
precipitant solution containing 10 mM Zinc Sulfate Heptahy-
drate, 100 mM MES buffer pH 6.5, and 25 % v/v polye-
thyleneglycol monomethyl ether 550. Crystals were cryopro-
tected with the mother liquor supplemented with 15% poly-
propylene glycol and immediately flash-frozen under a
stream of nitrogen.
875–886
X-ray crystallography. Crystals were grown by hanging-drop
vapor diffusion, and datasets were collected at the synchro-
tron Soleil (PROXIMA-1). Data were treated by XDS and
XSCALE (Kabsch, 2010). The structure was determined by
molecular replacement with Molrep (Vagin and Teplyakov,
2010) using the apo RBP structure as a search model (Farenc
et al., 2014). The crystals belong to the cubic space group
P213 with unit cell dimension of a = b = c = 165.4 Å, and a Vm
of 4.7 with 74% solvent for two monomers in the asymetric
unit.
They diffracted to a resolution of 2.1 Å (Table 1). Each of the
two monomers in the asymmetric unit is part of a trimer that
can be reconstituted by the threefold crystallographic axis.
Refinement was performed with AutoBUSTER (Blanc et al.,
2004) alternated with manual reconstruction with Coot
(Emsley et al., 2010). TriS was built from monosaccharides
and regularised with Coot (Emsley et al., 2010). The omit map
was calculated after four cycles of Cartesian simulated
annealing using Phenix (Adams et al., 2010). Figures were
made with the molecular graphics programme Pymol (Pymol,

2014).
Molecular modelling. The hexasaccharide was modelled by
extension of the trisaccharide. The coordinates of the extra
residues were obtained from the trisaccharide itself or from
high-resolution structures of protein–saccharide complexes
in the Protein Data Bank (PDB). A unique Galf structure
was found from the entry 2VK2. The TriS molecule was
generated by the CCP4 building option ‘Sketcher’ according
to standard bond lengths. The resulting structure was
docked ‘manually’ (by rotation-translation of the whole mol-
ecule and rotation of the dihedral angles) into the phage
1358 RBP receptor-binding groove using Coot (Emsley
et al., 2010). The resulting complex was idealised using
REFMAC5 (Murshudov et al., 2011) in refinement mode in
order to obtain a reasonable and clashless model as well as
favourable contacts. Figures were made with the molecular
graphics programme Pymol (Pymol, 2014).
Phages adsorption to L. lactis strains
Phage adsorption assays were performed as described pre-
viously (Sanders and Klaenhammer, 1980) with the following
modifications. One hundred microlitres of phage (104 pfu ml−1)
were mixed with 900 μl of bacteria (OD600 of 0.6 to 0.8). After
incubation at 30°C for 10 min, the mixture was centrifuged at
16,000 × g for 1 min. The supernatant was then titrated. The
percentage of adsorption was calculated with the formula:
100 × ((phage titer in adsorption assay without bacteria –
phage titer in supernatant after adsorption assay) / Phage titer
in adsorption assay without bacteria). All the assays were
performed in triplicates.
Accession numbers
X-ray structure and structure factors were deposited in the

Protein Data Bank with the ID code 4RGA.
Conflict of interest
None
Funding
This work was supported by a grant from the Agence Nation-
ale de la Recherche (grants ANR-11-BSV8-004-01) and
grants from Science Foundation Ireland (Grants 08/RSC/
B1393, 08/In.1/B2067, and 13/IA/1959). SM acknowledges
funding from NSERC of Canada (Strategic program). SM
holds a Tier 1 Canada Research Chair in Bacteriophages.
Acknowledgements
We thank the synchroron Soleil (Saint-Aubin, France) for
beam time allocation, and the staff of Proxima 1 beamline for
their assistance.
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875–886
RUBRIC FOR INQUIRY AND PROBLEM SOLVING
ASSIGNMENT
(Research Paper)
Dimensions Proficient – 2.0 Competent – 1.5 Developing –
1.0

Novice – 0.5
Framing the Issues
Identifies and/or addresses
questions problems and/or
hypothesis informed by
knowledge of context.
(Claim/Introduction)
Clearly frames and
addresses a research
question, hypothesis and/or
problem.
Demonstrates strong
knowledge of relevant
context.
Sufficiently frames and/or
addresses a research
question, hypothesis and/or

problem.
Demonstrates substantial
context.
Begins to frame and/or
address a research
question, hypothesis and/
or problem.
Demonstrates some
context.
Demonstrates limited or no
ability to frame or address
a research question,
hypothesis and/or problem.
Demonstrates little
context.

Evidence Gathering
Assembles, reviews and
synthesizes evidence from
diverse sources of relevant
knowledge.
(Methods)
synthesizes pertinent
information from many
relevant and appropriate
sources with diverse
points-of-view.
selects
pertinent information from
relevant and appropriate
sources with diverse

points-of-view.
Reviews information from
some relevant sources with
similar points-of-view.
Reviews limited
information
from few relevant sources
with limited points of
view.
Analysis
Uses evidence to address
questions, test hypotheses
and evaluate claims and
solutions.
(Results)
Clearly analyzes, evaluates
and organizes evidence to
support hypotheses, claims

and solutions.
Consistently analyzes,
evaluates and organizes
evidence to support
hypotheses, claims and
solutions.
Partially analyzes,
evaluates
and organizes evidence to
support hypotheses, claims
and solutions.
Attempts to analyze,
evaluate and organize
evidence to support
hypotheses, claims and
solutions.
Conclusions
Draws conclusions

supported by evidence;
identifies implications and
limitations.
(Discussion)
Citation of sources of
evidence
Cites referenced
papers/databases
throughout the text and in
the end section
(References in the APA
format)
Draws logical conclusions,
offers insightful solutions
strongly supported by
evidence.

Discusses limitations and
implications.
Correctly lists all
references cited in the text
in the APA format.
Draws logical conclusions,
offers solutions supported
by evidence.
Discusses limitations and
implications.
Lists all references but
there are some minor
errors and inconsistency.
Draws somewhat logical
conclusions, offers some

solutions supported by
some evidence.
Identifies some limitations
and implications.
Lists some but not all
references (either missing
or mixed in different
formats) in the Reference
section.
Attempts to draw
conclusions, offers few
solutions supported by
evidence.
Identifies few or no
limitations and
implications.
Does not list any reference

or lists them in a different
format.
1
Research Paper Assignment: NSF101 (about 3-4 pages)
Think of a topic that resonates with “My Major Connection to
Biology” or a
topic that “interested you recently on a disease/physical trait
after you heard
discussions on a news channel/social media" or a topic that you
think is often
"misunderstood by non-scientists".
Examples of Topics (you are free to choose your own different
topic also):
1. Genetically Modified Organisms (GMOs)
2. Climate Change and Cellular Respiration
3. Stem Cell Therapy
4. Vaccines
5. Gene Editing Technology
Competency: Inquiry and Problem Solving; Abilities: Written
and Digital

Inquiry is a systematic process of exploring issues or questions
through the collection and
analysis of evidence that results in informed conclusions or
judgments. Problem solving refers
to the ability to design, evaluate, and implement a strategy or
strategies to answer an open-
ended question, overcome an obstacle, or achieve a desired
goal. Analysis is the process of
breaking complex topics or issues into parts to gain better
understanding, often through
processes of revision, rethinking, and reorganization, to
advance a claim hypothesis, or
solution. Inquiry, analysis and problem-solving combine to form
a habit of mind critical to
academic and career advancement, thoughtful citizenship, and
sustained, life-long learning.
Important instructions and guidelines regarding the assignment
I. Final Draft- due by November 25th, 2019 (Monday). You
should submit your first draft by 11/11/19
(on ePortfolio) to get feedback for improvisation.
II. Total number of points possible – 10 points (will account for
15% of your total course grade). You
should deposit/upload your assignment on ePortfolio (See
instructions below. SSM will also help you).
For a tutorial on how to deposit student work, go to:
http://eportfolio.lagcc.cuny.edu/support/tutorials.htm and find

the section called, “Assessment for
Students.” Click on the adobe flash button for “Depositing
Assessment Artifact in Digication: Instructions
for Students.” You will see a brief video on how to deposit.
http://eportfolio.lagcc.cuny.edu/support/tutorials.htm
2
III. How to find background material to write the research
paper? Once you have decided a topic and
you want to find previous research done on it, you can use the
following resources:
A) Go to LAGCC Library webpage and click on Library Media
Resource Center. Here, you can find books
and online articles and even databases on your topic. You could
also schedule a meeting with a librarian
here to help you with your research on the topic. Detailed
Instructions will be covered in the 10/07/19
Library session.
B) Use the link (http://www.ncbi.nlm.nih.gov/pubmed) to find
and download some freely available
articles published on your topic in highly prestigious scientific

journals.
IV. Specific instructions on how to prepare the Research paper
A) Type the research paper in 12-point font, Times New Roman.
Include your Name, Major and Date of
submission, Professor’s Name and Course in the center of the
first page. Title your Research paper.
Include at least 2-3 journal or book citations in APA format
towards the end in the Reference section.
The total length of the paper should not exceed 3-4 pages, and it
should be double-spaced. Grammar
and Spelling does count. Please proofread your paper before you
submit.
B) The Research paper should have the following sections (in
this order):
a. Title (Topic Statement/Question being addressed)
b. Claim (Write a brief hypothesis on your topic-related
question you want to address)
c. Introduction (Background on the topic from a Biology angle.
You should try and cite
some references here also besides the next sections)
d. Methods or Experimental Procedures (Methods used in the
studies cited)

e. Results (Major findings of the studies you cited)
f. Discussion [Discuss here what is your final opinion on this
topic after reading the
different studies you cited and what you think is the
significance/implications of the
research carried out (It’s relevance to society, economy); you
could even discuss if you
thought there were some caveats in the studies and can suggest
some future avenues
that can be pursued]
g. References (Cite the references in APA format)
V. Please check the sample research papers and rubric for this
assignment on the course ePortfolio
(NSF 101_Dr. Richa Gupta Fall 2019).

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