Bacteriophage endolysin Lyt μ1/6: characterization of the C-terminal binding domain

R E S EA RCH L E T T E R

Bacteriophage endolysin Lyt μ1/6: characterization of theC-terminal binding domain

Lenka Ti�sakova1, Barbora Vidova1, Jarmila Farka�sovska1 & Andrej Godany1,2

1Department of Genomics and Biotechnology, Laboratory of Prokaryotic Biology, Institute of Molecular Biology Slovak Academy of Sciences (IMB

SAS), Bratislava, Slovakia; and 2Faculty of Natural Sciences, Department of Biotechnology, University of Ss. Cyril and Methodius in Trnava, Trnava,

Slovakia

Correspondence: Lenka Ti�s�akov�a, Institute

of Molecular Biology Slovak Academy of

Sciences (IMB SAS), D�ubravsk�a cesta 21,

SK-84551 Bratislava, Slovakia.

Tel.: +421 2 59307432;

fax: +421 2 59307416;

e-mail: [email protected]

Received 1 October 2013; revised 14

November 2013; accepted 14 November

2013. Final version published online 6

December 2013.

DOI: 10.1111/1574-6968.12338

Editor: Richard Calendar

Keywords

actinophage l1/6; Streptomyces

aureofaciens; in silico analysis; binding;

truncations.

Abstract

The gene product of orf50 from actinophage l1/6 of Streptomyces aureofaciens

is a putative endolysin, Lyt l1/6. It has a two-domain modular structure, con-

sisting of an N-terminal catalytic and a C-terminal cell wall binding domain

(CBD). Comparative analysis of Streptomyces phage endolysins revealed that

they all have a modular structure and contain functional C-terminal domains

with conserved amino acids, probably associated with their binding function.

A BLAST analysis of Lyt l1/6 in conjunction with secondary and tertiary struc-

ture prediction disclosed the presence of a PG_binding_1 domain within the

CBD. The sequence of the C-terminal domain of lyt l1/6 and truncated forms

of it were cloned and expressed in Escherichia coli. The ability of these CBD

variants fused to GFP to bind to the surface of S. aureofaciens NMU was

shown by specific binding assays.

Introduction

Endolysins are highly evolved enzymes encoded in bacte-

riophage genomes which are used to digest the bacterial

cell wall ‘from within’ at the terminal stage of the phage

multiplication cycle (Loessner, 2005). Endolysins from

phages infecting Gram-negative bacteria are mostly sin-

gle-domain globular proteins, whereas endolysins from

phages infecting Gram-positive bacteria feature a modular

organization, in which the enzymatically active domain

(EAD) is typically situated at the N-terminus and the cell

wall binding domain (CBD) at the C-terminus (Loessner,

2005; Hermoso et al., 2007; Fischetti, 2010).

In general, CBDs bind noncovalently to the unique car-

bohydrate components of the host cell wall peptidoglycan

and feature rapid binding kinetics, high affinity, and

extraordinary specificity. Fusion of a bacteriophage endoly-

sin CBD with GFP produces a fluorescent, heterologous

fusion product that is able to rapidly recognize and bind to

the host cells of a given species or even to individual sero-

vars (Schmelcher et al., 2011). Such protein constructs

have several uses, including visualizing the binding effi-

ciency of a GFP reporter gene or replacing a deleted EAD

with GFP, allowing GFP to be used as a spacer (Kornd€orfer

et al., 2006). Fluorescence is a highly desirable property

when developing methods for detecting bacterial cells or

when attempting to produce a protein with high binding

affinity, as is done, for example, by modular engineering

(Kretzer et al., 2007; Schmelcher et al., 2010).

The Gram-positive genus Streptomyces is widely used in

genetic research into antibiotic production, bacterial

physiology, and cell differentiation (Hopwood, 2007).

Actinophages infecting Streptomyces provide a source of

new genes for the organism, and this feature makes them

a very convenient tool for the genetic engineering and

characterization of Streptomyces (Hopwood, 2007; Maleki

& Mashinchian, 2011). The whole dsDNA of the actino-

phage l1/6 genome has been sequenced (GenBank

FEMS Microbiol Lett 350 (2014) 199–208 ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

MIC

ROBI

OLO

GY

LET

TER

S

DQ372923) and analyzed (Farka�sovska et al., 2007). The

1182-bp endolysin lyt l1/6 gene (GenBank AY321539.1)

from the ‘lysis cassette’ encodes a 393-amino acid protein

(Lyt l1/6) and is responsible for host cell lysis. Like the

endolysins from other phages which infect Gram-positive

bacteria, Lyt l1/6 has been shown to consist of an

N-terminal EAD and a C-terminal CBD (Farka�sovska

et al., 2003).

The aim of this study was to characterize the C-terminal

domain of endolysin Lyt l1/6, both bioinformatically and

experimentally. The bioinformatics approach involved

comparing all the Streptomyces phage endolysins available

in the public databases to identify their functional

domains and to locate any conserved amino acids which

may be responsible for their binding function. The

results of this comparison would then be used to con-

struct heterologous fusion proteins to verify whether these

conserved residues actually were responsible for the

unique cell wall binding properties of Lyt l1/6 CBD.

Experimentally, this would be carried out by producing

fusion proteins consisting of the Lyt l1/6 CBD or trun-

cated forms of it fused with GFP. These fusion proteins

would then be used in binding assays with S. aureofaciens

cells to demonstrate the predicted binding properties of

CBD to the cell wall peptidoglycan.

Material and methods

Bioinformatics analysis

Analysis of Streptomyces phage endolysins

The number of Streptomyces genomes available to date

(November 2013) was taken from the Entrez Genomes

viral database (Bao et al., 2004), and the nucleotide and

protein sequences of the Streptomyces phage endolysins

were retrieved from GenBank (Benson et al., 2013). Fur-

ther information was acquired from Uniprot (Apweiler

et al., 2004), the identity of each sequence was verified,

and appropriate sequences were retrieved. Protein

sequences were analyzed for functional domains using

CDD (Marchler-Bauer et al., 2013) and PFAM (Finn

et al., 2008). Both protein and nucleotide sequences were

then aligned using CLUSTALW2 (Larkin et al., 2007) on the

EBI server. The resulting alignments were manually col-

ored in MS-Word to indicate either invariant or con-

served residues. WebLogo (Crooks et al., 2004) was used

to prepare a graphical representation of the amino-acid

multiple sequence alignment (Fig. 1c).

To determine which amino acids within the Lyt l1/6CBD PG_binding_1 domain are conserved, a PROTEIN

PSI-BLAST analysis (Altschul et al., 1997) against only

the streptomycete phage and prophage endolysins and

other similar proteins was performed, with the CBD

sequence of Lyt l1/6 used as a query. To limit the num-

ber of sequences to be analyzed, only those from Strepto-

myces and their phages and those with > 50% identity

were examined. The relevant protein sequences were

retrieved, aligned in CLUSTALW2 and further analyzed. They

may be seen in Fig. 1b, with identical positions or closely

conserved residues colored appropriately.

Analysis of the endolysin Lyt l1/6 modularstructure

The secondary structure of CBD was predicted using the

SWISS-MODEL server (Arnold et al., 2006) via the

ExPASy web portal (Artimo et al., 2012). The target was

the PG_binding_1 domain identified by CDD. The 3D

structure and function of CBD were predicted using the

I-TASSER server (Zhang, 2008).

Experimental part

Bacterial strains, vectors, media, and growthconditions

The bacterial strains used in this study were E. coli MC1061

(Casadaban & Cohen, 1980) for cloning experiments,

E. coli BL21(DE3) (Stratagene) as a host for the expression

of recombinant proteins and S. aureofaciens NMU (Far-

ka�sovska et al., 2003) for preparation of substrate for bind-

ing assays. The E. coli strains were grown at 37 °C in

Luria–Bertani medium (Sambrook & Russel, 2001), supple-

mented with 100 lg mL�1 ampicillin where necessary.

The S. aureofaciens NMU strain was grown at 30 °C in

medium 16 (in g L�1: agar 25, dextrin 15, peptone 5,

sucrose 3, yeast extract 1, yeast extract 1; in mg L�1:

NaCl 50, K2HPO4 50, MgSO4, pH 7.2, urea 10) for its

propagation and in nutrient broth no.1 (Serva, Germany)

for binding assays.

Vectors pET-21a(+) and pET-21d(+) (Novagen, Germany)

were used for cloning and expression of the Lyt l1/6 gene,

the CBD, and its truncations in E. coli. Vector pET28-gfp, a

gift from Dr. Bukovska (IMB SAS, Bratislava), was used for

the PCR amplification of gfp.

General DNA techniques and construction ofplasmids

Standard DNA manipulations were performed essentially

as described by Sambrook & Russel (2001). The purified

genomic DNA of actinophage l1/6 (Farka�sovska et al.,

2003) was used as a template for the PCR amplification of

Lyt l1/6 and its C-terminal regions. The CBD gene

sequence was used as a template for the PCR amplification

FEMS Microbiol Lett 350 (2014) 199–208ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

200 L. Ti�s�akov�a et al.

of the CBD truncations (Table 1). PCR amplifications

were performed using Dream Taq polymerase (Thermo

Scientific). Gene sequences were amplified using PCR

primers (Table 1) with HindIII and either NdeI or NcoI

recognition sites to introduce appropriate restriction

enzyme sites for subcloning into pET21a or pET21d vec-

tors. PCR products were purified using a GeneJet PCR

Purification Kit (Thermo Scientific), digested with appro-

priate restriction enzymes, and introduced to expression

vectors pET21a+ or pET21d+ using a Rapid Ligation Kit

(Thermo Scientific). The gfp coding sequence from tem-

plate pET28-gfp was PCR amplified using primers GFP-f

and GFP-r (Table 1). The gfp PCR fragment was digested

with HindIII–XhoI endonucleases and subcloned into the

corresponding HindIII-XhoI sites of all CBD constructs.

For pET21a-gfp, an appropriately digested gfp insert was

introduced directly into the corresponding HindIII-XhoI

site of a pET21a+ vector. Correct sequences of all

constructs were verified by sequencing. Escherichia coli

BL21 (DE3) competent cells were then transformed with

the recombinant plasmids for protein expression with a

C-terminal His6Tag.

Overexpression and partial purification of the Lytl1/6 CBD gene products

Escherichia coli BL21(DE3) cells harboring the recombi-

nant plasmids (Table 1) were grown overnight in LB

(a)

(b)Q7Y4H8 214 YQTTINGLAYGYGAQGDQVTAVGRALVAHGFGSHYQQGPGPNWTDADTENYADYQRSLGYTGQAADGVPGSDSLRQLLGTLPGGRTVSLAHVIAAAQADPPAAQGHQTYGPDVQIVEQA---LADEGLLDQQWVDG-SFGSRTVSAYAAWQRRCGYSGSGADGIPGKASLDRLAAAHGFTTTD 393D7NW66 182 -------------------------------------TPKP--------------------------------------------VIDLSKVVTAARTNPPMAKRTVTY-AGVADVKAW---LIAEGLLVKSDTDG-HFGQRVLDAYKAWQRRCGYSGAAADGVPGMTSLRKLAVKHGRTVTA 278K4IB87 179 YQVTINGLKYGYGAQGSHVTTVGKALVAKGFGKHYAEGPGPTWSDADTLNYADFQRSLGYSGSDADGVPGEGSLKTLLGSLPGASAPAPAAKPAAKKYEPFPGASFFKRAPKSAIVTAMGKRLVAEGCGVYSSGPGPQWTESDRKSYAKWQRKLGYTGSAADGYPGKASWDKLHVPEV----- 356K4I2E3 180 YQVTINGLKYGYDAYGDHVTKVGQALVAKGHGDHYASGPGPRWTDADTLNYADFQRSLGYSGADADGVPGESSLRALLGYLPGATATV--------KYEPFPGATFFKNAPRSAIVAAMGKRLVAEGCSAYSSGPGPQWTEADRLSYQKWQRKLGYSGADADGWPGKTSWDKLRVPEV----- 350K4HYE4 184 MDSMRDRVDARLD-----------------------DKPKPKPPAPKPTPPAKPKPTPPKKP-----------------------AVSLARLITAARIDPAKKGTPVSYAGARVVEQAL----AAEGLLDRALIDG-HFGTATRTAYGRWQARQGYRGTAADGVPGRASLGALAARHGFTVTA 315K4IBM9 139 KRARRDPYLYGYG---------------------YPDVAGSLSADPDASRFGYKHASTSKPAVAPKPKPKPKPKP--------------------KAYEPFPGAAFFKREPKSAIVTAMGKRLVAVGCSAYKSGPGPQWTDADRASYAKWQRKRGYSGADADGWPGKASWDALKVPKV----- 276

. . :* . * * : :* ** : ** *: *** ** :* * . .

(c)

Fig. 1. Modular organization of Lyt l1/6 and its CBD truncations (visualized by CLC Main Workbench, Aarhus, Denmark). Numbers above the

scheme are for amino acids (a) Lyt l1/6 consists of an N-terminal catalytic domain (M1 to N199) and a C-terminal binding domain (Y214 to

D393), which are connected with a proline-rich linker. Sequences of the Lyt l1/6 C-terminal domain truncations: CBD (Y214 to D393), CBD-BS

(Y264 to D393), CBD-PG (T321 to D393), and CBD-3H (P324 to A386). The gene for GFP was subcloned into the C-terminus of these

sequences, in the expression and cloning vector pET21a(d) with a C-terminal His6Tag. In all cases, the expressed fusion products showed binding

activity toward Streptomyces aureofaciens. (b) C-terminal conservation of amino-acid residues of the six available identified Streptomyces phage

endolysins: Q7Y4H8 from Lyt l1/6, D7NW66 from phiSASD1, K4IB87 from R4, K4I2E3 from phiHau3, K4HYE4 from SV1, and K4IBM9 from

TG1. CLUSTALW2 alignment of representative endolysin protein sequences. Stars = identity; colon = highly conserved amino acid side chain; single

dot = weakly conserved amino acid side chain; bold and italic = highlighting of the PG_binding_1 domain (P324 to D393) within the C-terminal

part of Lyt l1/6. (c) WebLogo of Streptomyces phage endolysin sequences showing the degree of amino-acid conservation and identity regarding

the Lyt l1/6 C-terminal domain. Amino acids belonging to the three a-helices of the Lyt l1/6 PG_binding_1 domain are underlined in the

sequence and marked above the alignment.


Lyt l1/6 cell wall binding domain 201

Table

1.Fusionconstructsan

dtheirprotein

productsshowingcharacterization,theoligonucleo

tides

usedforPC

Ram

plificationofthelytinserts,

therationaleforeach

construct’s

designan

d

theactual

bindingactivity

offusionproteinsin

bindingassay.

Theintensity

ofbindingto

thestreptomycetecellwallsismarkedwith+fortheweakest

fluorescen

ce,whichwas

notobserved

,++

forweakfluorescen

ce,+++formed

ium

fluorescen

ce,++++forthestrongest(�

forno)fluorescen

ceunder

fluorescen

cemicroscopyconditions

Constructs

Fusionprotein

products

Nam

e

Prim

ersusedfortheam

plificationoflytinserts

(withrestrictionsitesincluded

)

Size

oflyt

insertsin

bp

Sequen

cerange

oflytinserts

Nam

e

Size

inkD

a

without

His-Tag

Rationale

Bindingactivity

to

thestreptomycete

substrate

pET21a-lyt-gfp*

Gp50-f:(NdeI)GGAATTCCATA

TGCCCGACTTG

TGGATG

CCTG

GTG

C**

Gp50-r:(HindIII)CCCAAGCTT

GTC

GGTG

GTG

GT

GAAGCCGTG

GGCGGC**

1180

V1–D393

LYT-GFP

69

Functionofwhole

Lytl1/6

=‘LYT’

Lysis

pET21a-lytCBD- gfp*

CBD-f:(NdeI)GGAATT

CATA

TGCGGTA

CCAGA

CCACCATC

AAC**


GTC

GGTG

GTG

GT

GAAGCCGTG

GGCGGC**

599

Y214–D

393

CBD-G

FP48.1

Functionofwhole

CBD

=‘CBD’

++++

pET21d-lytCBD-BS-gfp*

BS-f:(NcoI)CATG

CCATG

GCATA

CGCCGACTA

C

CAGCGG**


GTC

GGTG

GTG

GT

GAAGCCGTG

GGCGGC**

389

Y264–D

393

BS-GFP

40.7

Truncationof

CBD

with

preserved

potential

binding

site

=‘BS’

++

pET21d-lytCBD-PG- gfp*

PG-f:(NcoI)CATG

CCATG

GCACCTA

CGGGCCC

GATG

TGC**


GTC

GGTG

GTG

GT

GAAGCCGTG

GGCGGC**

217

T329–D

393

PG-G

FP34.5

TruncationofCBD

withwhole

PG_b

inding_1

domain=‘PG’

++

pET21d-lytCBD-3H- gfp*

3H-f:(NcoI)CATG

CCATG

GCACCCGATG

TGCA

GATC

GTG

**

3H-r:(HindIII)CCCAAGCTT

GGCGGCGGCGAGGCG**

186

P324–A

386

3H-G

FP33.4

Truncationof

CBD

withonly

threehelices

of

PG_b

inding_

domain=‘3H’

+++

pET21a-gfp

GFP-f:(HindIII)CCCAAGCTT

ATG

AGTA

AAGGAG

AAGAA**

GFP-r:(XhoI)CCGCTC

GAGTTTG

TATA

GTTCATC

CAT*

*

715

�GFP

26.5

Neg

ativecontrol

forbinding

assays

=GFP

�

*Su

bcloningofthegen

eforgfp

isunderlined

.

**Restrictionsites(in

italics)

areunderlined

.



medium containing ampicillin (100 lg mL�1). The cul-

ture was diluted into fresh medium and grown under

shaking at 37 °C to an OD600 nm of 0.5. Expression of

the CBD gene was induced by addition of isopropyl-b-D-thiogalactopyranoside (IPTG) to a final concentration

of 0.7 mM followed by overnight incubation. Cells were

harvested from 100 mL cultures (2000 g, 10 min,

4 °C), and the pellet was resuspended in BugBuster

MasterMix (Novagen) supplemented with ProteoBlock

Protease Inhibitor Cocktail (Thermo Scientific) and

incubated at room temperature for 30 min. The soluble

protein fraction was separated from the cell debris by

centrifugation (11 000 g, 15 min, 4 °C). The cleared

supernatant was applied to a nickel-nitrilotriacetic acid

(Ni-NTA) Agarose (Qiagen, Germany) in a slurry and

mixed gently for 40 min at 4 °C. Proteins were eluted

with 50 mM NaH2PO4, 300 mM NaCl, and 250 mM

imidazole. Buffer exchange and protein concentration

were performed using Amicon� Ultra-4 Centrifugal Fil-

ter Units (30 kDa) (Merck Millipore, Germany). All

samples were either converted to storage buffer

(50 mM Tris-HCl, pH 8.0 + 1 mM dithiothreitol) or

assayed directly for purity via sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE) (Sup-

porting Information, Fig. S1).

SDS-PAGE was performed on a 12% gel using a Spec-

tra Multicolor Broad Range Ladder (Thermo Scientific)

as the molecular size marker. Gels were stained in Coo-

massie stain for 15 min and then destained via conven-

tional methods. Proteins were stored at �20 °C until

assayed.

Binding activity assays and fluorescencemicroscopy

The specific binding of fusion proteins (Table 1) to

S. aureofaciens NMU cells was performed according to

Loessner et al. (2002) with some modifications. Cells

(16 h of growth) were harvested by centrifugation, resus-

pended in 1/10 volume of Tris-HCl (50 mM, pH 8.0).

Subsequently, 100 lL cells and 50 lL of either fusion

protein or a GFP negative control were mixed and incu-

bated at room temperature for either 5 min (CBD-GFP

and CBD-GFP) or 45 min (BS-GFP, PG-GFP and

3H-GFP). Cells were centrifuged and the supernatant

discarded. The cells were then washed twice in 500 lLof Tris-HCl buffer, and the pellet was resuspended in

50 lL Tris-HCl. Cells were visualized on poly-L-lysine-

treated slides using fluorescence microscopy. All images

were obtained using a LEICA DM2500 microscope

equipped with a LEICA DFC290 HD camera and LAS

software.

Results and discussion

Analysis of Streptomyces phage endolysins and

their modular structure

Presently (November 2013), GenBank contains the com-

plete nucleotide sequences of nine Streptomyces bacterio-

phages, including ΦC31 (Accession: NC_001978), ΦBT-1(Accession: NC_004664), l1/6 (Accession: NC_007967.1),

VWB (Accession: NC_005345), ΦSASD1 (Accession:

NC_014229), ΦHAU3 (Accession: JX182369), R4 (Acces-

sion: JX182370), SV1 (Accession: JX182371), and TG1

(Accession: JX182372). Among them, six putative endoly-

sins have been identified (Table 2). All of these endolysins

possess typical Gram-positive two-domain organization.

Lyt l1/6 has the longest sequence among all available

endolysins, and its encoding gene is located at the very

end of the l1/6 genome as a part of the late transcribed

genes (data not shown).

The streptomycete endolysin CBDs contain either a

putative peptidoglycan-binding domain (PG_binding_1)

or a peptidoglycan recognition protein (PGRP) domain.

The peptidoglycan-binding domain has a common core

structure consisting of three alpha helices (Dideberg et al.,

1982) and has been shown experimentally to bind pepti-

doglycan (Briers et al., 2007). It has been found at the

N- or C-terminus of a variety of enzymes involved in

bacterial cell wall degradation (Foster, 1991). Many pro-

teins possessing this domain have not yet been character-

ized, including the Streptomyces phage endolysins. PGRPs

have at least one carboxy-terminal PGRP domain (c. 165

amino acids long), which is homologous to bacteriophage

and bacterial type 2 amidases (Dziarski & Gupta, 2006).

We focused on the peptidoglycan-binding domain as it is

present in Lyt l1/6.All Streptomyces phage endolysin sequences contained

16 invariant amino acids and 12 other conserved posi-

tions (Fig. 1b). Nearly all identical amino acids were

found at the protein C-termini, where the binding prop-

erties of Gram-positive modular endolysins are most

often located. These conserved regions are graphically

depicted in Fig. 1c as the overall height of a stack which

indicates the sequence conservation at a given position.

The height of the symbols within each stack indicates the

relative frequency of each amino acid at that position.

The final two predicted helices in all six aligned sequences

contain five identical residues: Y357, W360 and Q361 in

helix2, and S379 and L383 in helix3 (Fig. 1b and c).

To emerge a clearer picture of the phage endolysins

CBDs against the C-terminal domain of Lyt l1/6, 33 of

those sequences found by the PROTEIN PSI-BLAST

analysis with > 50% sequence identity were chosen for



Table

2.Th

elistofputative

endolysinsfrom

Streptomyces

phag

esfoundin

Gen

Ban

k(NCBIsearch

–keyw

ords‘phag

een

dolysin’an

d‘strep

tomyces’;verified

inUniProt).Included

features:

host

(labhost),references,

datab

aseIDs,

gen

omic

rangean

dgen

elength,protein

massan

dlength,an

diden

tified

conserved

domains;

nt–nucleo

tides;aa

–am

inoacids

Streptomyces

Phag

eHost

EndolysinID

Sequen

ce

length

Mass(Da)

Gen

omic

range

Conserved

domains

RefSeq

Uniprot

nt

aaCatalytic

Binding

l1/6

S.au

reofacien

sYP_579222.1

NC_0

07967.1

Q7Y4H8

1182

393

42132

36463–3

7644

–pfam01471

PG_b

inding_1

ΦSA

SD1

S.avermitilis

YP_003714747.1

NC_0

14229.1

D7NW66

837

278

29600

16596–1

7432

–pfam01471

PG_b

inding_1

R4

S.coelicolorA3(2)

YP_006990140.1

NC_0

19414.1

K4IB87

1071

356

37084

21877–2

2947

Amidase_domain.

N-acetylm

uramoyl- L-alanine

amidaseactivity

cd06583

PGRP

ΦHau

3S.

coelicolorA3(2)

YP_006906203.1

NC_0

18836.1

K4I2E3

1053

350

37152

21373–2

2425

Amidase_domain.

N-acetylm

uramoyl- L-alanine

amidaseactivity

cd06583

PGRP

SV1

S.venezuelae

YP_006906963.1

NC_0

18848.1

K4HYE4

948

315

33449

16368–1

7315

–COG3409

Putative

pep

tidoglycan-binding

domain-containingprotein

cd06583

PGRP

pfam01471

PG_b

inding_1

TG1

S.avermitilis

YP_006907195.1

NC_0

18853.1

K4IBM9

831

276

29818

15771–1

6601

cl11438

NLPC_P60;NlpC/P60family

(unkn

ownfunction)

PG_b

inding_1

=Pu

tative

pep

tidoglycanbindingdomain;PG

RP=Peptidoglycanrecognitionproteins(PGRPs).



extended analysis against the Streptomyces phage and pro-

phage endolysins. Although the alignment showed less

overall amino-acid conservation (Fig. 2) than the six iden-

tified Streptomyces phage endolysins (Fig. 1c), a similar

degree of conservation can be observed at their C-termini;

specifically, G365, A371, G373, and L383 (Lyt l1/6 num-

bering) were all invariant (Fig. 2). This conservation sug-

gests that there is likely to be a link between these

particular amino acids and the binding function of the

C-terminal modules. In this connection, it is worth noting

that the greatest level of conservation in all the Streptomy-

ces phage endolysin alignments in this study is observed

in exactly the region encoding the three a-helices of

the putative PG_binding_1 domain within the Lyt l1/6C-terminal domain.

Analysis of the endolysin Lyt l1/6 modular

structure

Previous bioinformatics analysis suggested that the endol-

ysin Lyt l1/6 is a modular protein composed of two

functional domains separated by a linker region rich in

proline residues (Fig. 1a) and that it possesses a putative

cell wall binding domain containing the PG_binding_1

domain in its C-terminal part (Farka�sovska et al., 2003),

but no further experiments were carried out to determine

its function. The supposed catalytic domain at its N-ter-

minus, which seems to be unrelated to any of the known

enzymatic domains of phage endolysins, will be examined

elsewhere. In this study, the presence of a PG_binding_1

domain was identified by CDD and confirmed by Protein

BLAST analysis. A SWISS-MODEL prediction of the sec-

ondary structure of CBD (the helices indicated in Fig. 1a

and c) was also quite useful because only a few templates

with very low sequence homology were available. The

bacterial PG_binding_1 domain is readily detected in

many different proteins, not only from Streptomyces, in

several domain databases. Its binding sites for streptomy-

cete peptidoglycan are still poorly understood.

According to the predicted 3D structure of Lyt l1/6CBD region by I-TASSER, Y357 and W360 occupied the

domain core, and Y357 interacted with the highly

Q7Y4H8 324 PDVQIVEQALAD--------EGLLDQQ--WVDGSFGSRTVSAYAAWQR-RCG--YSGSGADGIPGKASLDRLAAAHGFTTTD 393D7NW66 209 AGVADVKAWLIA--------EGLLVKS--DTDGHFGQRVLDAYKAWQR-RCG--YSGAAADGVPGMTSLRKLAVKHGRTVTA 278K4IB87 288 PKSAIVTAMGKR-----LVAEGCGVYSS-GPGPQWTESDRKSYAKWQR-KLG--YTGSAADGYPGKASWDKLHVPEV----- 356K4I2E3 282 PRSAIVAAMGKR-----LVAEGCSAYSS-GPGPQWTEADRLSYQKWQR-KLG--YSGADADGWPGKTSWDKLRVPEV----- 350K4HYE4 238 KKGTPVSYAGARVVEQALAAEGLLDRAL-IDG-HFGTATRTAYGRWQA-RQG--YRGTAADGVPGRASLGALAARHGFTVTA 315K4IBM9 207 PKSAIVTAMGKR-----LVAVGCSAYKS-GPGPQWTDADRASYAKWQR-KRG--YSGADADGWPGKASWDALKVPKV----- 276G8XHL4 491 -HPDDVFTVELA-----LVDEGLLDREW-A-DGSFGTRTITAYAELQR-RYG--YSGQMADGIPGTESLTRLGRAHGFTVR- 561F8JK13 499 -HPDDVFTVELA-----LVDEGLLDREW-A-DGSFGTRTITAYAELQR-RYG--YSGQMADGIPGTESLTRLGRAHGFTVR- 569I7A9I3 288 PKSAIVTAMGKR-----LVAEGCGVYSS-GPGPQWTESDRKSYAKWQR-KLG--YTGSAADGYPGKASWDKLHVPEV----- 356Q9ZX99 294 PKSPIVTAMGKR-----LVAEGCSAYRS-GPGAQWTNADKASYAKWQR-KRG--YSGADADGWPGKTTWDALKVPKV----- 362L1L7W7 332 TDVRLVEEALAA--------EGLLERG--YVDGSFGTRTIEAYAAWQRSRAGGSYRGRDADGVPGRASLTRLGDRHGFTVIA 404L7ETE1 202 -FPADVRPVEAA-----LVAEGLLDPTF-GGDGSFGSHTVDAYAAFQR-QQG--FTGANADGIPGESTLAALGSRHGFTVAA 274H0BPX9 234 ADVKIVEAALQK--------EGLLGASY-AKDGSFGSLTRAAYSAWQR-RCG--YSGSAADGIPGKASLEKLGVKRGFKVKA 304I2MXW1 234 RPGTPVSYPGVKTVEKALVKEGLLTAGL-ADG-HYGTATKDAYAAWQR-RLG--YTGGAADGIPGQASLKKLATKHGFTVTP 310K1UV81 284 ADVKIVEAALKA--------EGLLAATY-AADGSWGTKTDTAYDAFRR-KMG--YTGSAATGSVGLESLKKLAARHGFTAKA 353I0CEJ4 242 RKSPIVTAMGRR-----LVAEGCGRYSQ-GPGPNWTNADKASYAAYQR-KLG--YSGAAADGIPGKTSWDKLRVPKQL---- 311D0UZA7 376 RKSPLITAMGRR-----LVAEGCGKYKQ-GPGPNWTNVDKASYSAWQR-KLG--YSGTAADGIPGKASWDKLRVPKQL---- 445D9W712 218 RDSEIVTAMGKR-----LVAEDCDHYQE-GPGPEWTDADQESYAAWQR-KLG--FSGDDADGIPGEVSWDKLRVPQD----- 286D9XZ47 379 AVNDQVTRLGEQ-----LVRKGFGRYYADGPGPRWSEADRRNVEAFQR-AQG--WRGGAADGYPGPETWRRLFLS------- 446D9W669 221 RESKIITAMGKR-----LVAEGCDRYEE-GPGPEWTDADKKSYAAWQR-KLG--YSGDDADGIPGKKSWDKLRVPNV----- 289E2PZH7 199 RRSPIVTAMGRR-----LVAEGCGRYEI-GPGPAWSEADRRSYAAWQR-KLG--YSGAAADGIPGKTSWDRLKVPNT----- 267G2P5J6 222 RQSKIITAMGKR-----LVAEGCGRYEE-GPSPEWTEADRKSYAAWQH-KLG--YSGDGADGVPGKASWDKLRVPNV----- 290F3Z9F2 215 PKSALVTAMGRR-----LVAEGCSAYAE-GPGPQWTAADRASYAKWQR-KLG--YTGADADGWPGAASWRALKVPGV----- 283D9UFT6 215 PKSALVTAMGRR-----LVAEGCSAYAE-GPGPQWTAADRTSYAKWQR-KLG--YTGPDADGWPGAASWRALKVPGV----- 283D6AD83 138 RNSAIVTAMGKR-----LVAEGCGRYTV-GPGPAWSEADRKSYAAWQR-KLG--YTGGDADGIPGKSSWDRLKVPNV----- 206G0Q9L7 223 RNSAVVTAMGRR-----LVSEGCGRYTV-GPGPAWSEADRKSYAAWQR-KLG--YTGGDADGIPGKSSWDRLKVPNV----- 291H0B7I0 180 RNSAVITAMGKR-----LVSEGCGRYTV-GPGPAWSEADRKSYAAWQR-KLG--YTGGDADGVPGKSSWDRLKVPNV----- 248B1VX31 223 RNSAVVTAMGRR-----LVSEGCGRYTV-GPGPAWSEADRTSYAAWQR-KLG--YTGGDADGIPGKSSWDRLKVPNV----- 291K4REH3 227 QKSPVITAMGRR-----LVAEGCGRYEE-GPGPEWTEADRRSYAAWQQ-KQG--FKGKDADGIPGRVTWERLKVPNGPN--- 297K4RC28 1 --MKTVEAALVA--------EDLLSKA--LADGHFG--TATAYAAWQR-RCG--WSIDDADGTPDLASLTELGKRRGFDVKE 65B1W438 240 PSSPVVTAMGRR-----LSAEGCGAYAV-GPGPRWTEADRRSYAAWQR-KLG--FRGAEADGWPGRTSWNALKVPYTTKSP- 313G0PXR4 240 PSSPVVTAMGRR-----LSAEGCGAYAV-GPGPRWTEADRRSYAAWQR-KLG--FRGAEADGWPGRTSWNALKVPYTTKSP- 313I2N4B2 230 PSSPVITAMGRR-----LLAEGCGEYAV-GPGPRWSEADRRSYARWQR-KLG--FRGADADGWPGAASWNALKVPHTP---- 299

: . . : : * : * * . : *

Fig. 2. CLUSTALW2 multiple sequence alignment of 33 Streptomyces phage and prophage protein sequences (UniProt IDs) with > 50% sequence

identity from PROTEIN PSI-BLAST. Conserved amino acids are highlighted. Stars = identity; colon = highly conserved amino acid side chain; single

dot = weakly conserved amino acid chain; bold = highlighting of the PG_binding_1 domain from Lyt l1/6 (P324 to D393).



conserved L383 of helix3. Y357 also interacted with a

conserved P375 found in the loop between helix2 and

helix3. W360, on the other hand, interacted with the con-

served residues L336 and E339 (Supporting Information,

Fig. S2). It appears therefore that both Y357 and W360

are the important building blocks needed to stabilize the

endolysin-binding domain architecture. The loop between

the second and the third helix is highly conserved and

rich in glycines. Much bigger conformational flexibility is

expected there, allowing a tight turn in the chain leading

to helix3. S379 and L383 in helix3 are, respectively,

invariant and highly conserved residues; both are fre-

quently found in protein functional centers. While the

leucine side chain is generally unreactive, it could play a

role in substrate recognition. In particular, hydrophobic

amino acids are often involved in the binding or recogni-

tion of hydrophobic ligands, which can occasionally be

associated with peptidoglycans (Betts & Russell, 2003).

These postulated roles of the conserved amino-acid resi-

dues will require additional experimental verification. In

the next part of the present study, the effects of trunca-

tions of the Lyt l1/6 C-terminal domain on binding to

Streptomyces cells are explored.

Binding activity and fluorescence of fusion

proteins

Fusion proteins were previously used to analyze the

contribution of the CBDs to the ability of the endolysins

they are found in to bind to their targets, most nota-

bly between clostridial and pneumococcal peptidogly-

can hydrolase enzymes and in heterologous fusions (Diaz

et al., 1990, 1991; Croux et al., 1993a, b; Loessner et al.,

2002; Mayer et al., 2011; Mao et al., 2013). Although not

always a necessity for baseline activity, some endolysins

require a cell binding domain to achieve high levels of

activity (Donovan et al., 2006; Sass & Bierbaum, 2007;

Rodriguez-Rubio et al., 2012). The in silico and in vitro

analyses reported here were aimed primarily at character-

izing the Lyt l1/6 C-terminal region from Y214 to D393,

which corresponds to its putative CBD. Although believed

to play a role in Streptomyces cell-wall surface-binding

specificity, ability to actually bind to the cell walls needs

to be empirically determined. Several truncations of the

C-terminal domain (Fig. 1a) were carried out to deter-

mine which part of the CBD sequence is responsible for

its binding activity. When designing the CBD truncations,

the I-TASSER tertiary structure prediction and the amino

acids predicted to be responsible for the CBD-binding

activity were taken into account, resulting in the produc-

tion of CBD-BS and CBD-PG. To determine whether the

in silico-identified PG_binding_1 domain within this CBD

actually corresponds with its predicted binding function,

CBD-3H was constructed.

Purified proteins from all C-terminal fusion constructs,

depicted in Fig. 1a, were tested in binding assays for their

ability to bind to S. aureofaciens cells; the ability of CBD-

GFP to bind to budding spores was also tested. Figure 3

shows that all five fusion proteins hold evident binding

activity, even though the incubation time of the binding

assays to Streptomyces cells needed to be increased for the

smaller proteins from 5 min (CBD-GFP and BS-GFP) to

45 min (PG-GFP and 3H-GFP). Cell wall binding of the

intact LYT-GFP could not be properly tested because of

its lytic activity. In all cases, no binding activity of

unfused GFP, used as a negative control, was detected

(Fig. 3). It must be emphasized that each and every

fusion protein expression and isolation resulted in a

slightly different intensity for the green colored proteins,

apparently independently of the protein concentration

used in the binding assays. It could be also presumed that

the CBD truncations differ in protein folding and that

(a)

(b)

1 2 3 4 5 6 7 8

Fig. 3. The binding activity assays of GFP-tagged LYT, CBD and its derivates BS, PG, and 3H to the cell surface of Streptomyces aureofaciens

NMU show that binding is detectible in all cases. The binding abilities of fusion proteins LYT-CBD (1), CBD-GFP (2, 3), BS-GFP (4), PG-GFP (5),

3H-GFP (6), and single GFP (7, 8), assayed and visualized by phase contrast (a) and fluorescence (b) microscopy, magnification 10009. (1)

Binding of LYT-CBD caused immediate cell lysis; binding of CBD-GFP to the cells (2) and budding spores (3); binding of the truncated fusion

products to the cells (4–6).



the larger GFP part of the fusion products could play a

significant role (Kornd€orfer et al., 2006). Nevertheless,

this assumption would still have to be empirically verified

in more detailed protein studies. In addition, to more

exactly identify the part of the CBD responsible for full

binding activity, several point mutants of the conserved

amino acids identified in this study should perhaps be

prepared for future studies.

In this study, we compared all identified Streptomyces

phage endolysins and found that their C-terminal regions

showed the most conservation, especially in helix2 and

helix3 of the putative PG_binding_1 domain, which con-

tained several invariant amino acids. These amino acids

might be involved in the binding or recognition of

ligands on the Streptomyces cell wall surface. Further, with

the help of in silico analyses of Lyt l1/6, we performed

several truncations of its C-terminal domain. Binding

assays of expressed fusion proteins of these truncations

demonstrated that CBD has the ability to direct endolysin

Lyt l1/6 to the S. aureofaciens cell surface when applied

exogenously. In conclusion, we report the development of

novel chimeric truncations of the C-terminal domain of

Lyt l1/6 with confirmed binding activities to S. aureofac-

iens cells. Furthermore, we demonstrated that the

PG_binding_1 domain within the CBD of Lyt l1/6 is

responsible for the binding. We expect that these Lyt l1/6 C-terminal binding domain constructs will serve as an

intermediate to be used in the preparation of chimeras

with other types of binding domains to detect and visual-

ize various Gram-positive bacterial species.

Acknowledgements

This research study was supported by funding from the

scientific grant agency of the Ministry of Education of the

Slovak Republic and the Slovak Academy of Sciences

(VEGA, Grant no. 2/0140/11). The fluorescent microscope

was financed from the project ‘The center of excellence for

utilization of information on bio-macromolecules in dis-

ease prevention and in improvement of quality of life’

(ITMS 26240120003) supported by the Research and

Development Operational Program funded by the ERDT.

In addition, we thank Dr. GabrielaBukovska (IMB SAS) for

providing the gfp template (pET28-gfp) for PCR amplifica-

tion and Dr. Jacob Bauer (IMB SAS) for critical reading of

the manuscript.

References

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z,

Miller W & Lipman DJ (1997) Gapped BLAST and

PSI-BLAST: a new generation of protein database search

programs. Nucleic Acids Res 25: 3389–3402.Apweiler R, Bairoch A, Wu CH et al. (2004) UniProt: the

Universal Protein knowledgebase. Nucleic Acids Res 32:

D115–D119.Arnold K, Bordoli L, Kopp J & Schwede T (2006) The

SWISS-MODEL Workspace: a web-based environment for

protein structure homology modelling. Bioinformatics 22:

195–201.Artimo P, Jonnalagedda M, Arnold K et al. (2012) ExPASy:

SIB bioinformatics resource portal. Nucleic Acids Res 40:

W597–W603.

Bao Y, Federhen S, Leipe D, Pham V, Resenchuk S, Rozanov

M, Tatusov R & Tatusova T (2004) National center for

biotechnology information viral genomes project. J Virol 78:

7291–7298.Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I,

Lipman DJ, Ostell J & Sayers EW (2013) GenBank. Nucleic

Acids Res 41: D36–D42.Betts MJ & Russell RB (2003) Amino acid properties and

consequences of substitutions. Bioinformatics for Geneticists

(Barnes MR & Gray IC, eds), pp. 289. John Wiley & Sons,

Ltd, Chichester, UK.

Briers Y, Volckaert G, Cornelissen A, Lagaert S, Michiels CW,

Hertveldt K & Lavigne R (2007) Muralytic activity and

modular structure of the endolysins of Pseudomonas

aeruginosa bacteriophages phiKZ and EL. Mol Microbiol 65:

1334–1344.Casadaban MJ & Cohen SN (1980) Analysis of gene control

signals by DNA fusion and cloning in Escherichia coli. J Mol

Biol 138: 179–207.Crooks GE, Hon G, Chandonia JM & Brenner SE (2004)

WebLogo: a sequence logo generator. Genome Res 14:

1188–1190.Croux C, Ronda C, Lopez R & Garcia JL (1993a) Interchange

of functional domains switches enzyme specificity:

construction of a chimeric pneumococcal-clostridial cell wall

lytic enzyme. Mol Microbiol 9: 1019–1025.Croux C, Ronda C, Lopez R & Garcia JL (1993b) Role of the

C-terminal domain of the lysozyme of Clostridium

acetobutylicum ATCC 824 in a chimeric pneumococcal–clostridial cell wall lytic enzyme. FEBS Lett 336:

111–114.Diaz E, Lopez R & Garcia JL (1990) Chimeric phage-bacterial

enzymes: a clue to the modular evolution of genes. PNAS

87: 8125–8129.Diaz E, Lopez R & Garcia JL (1991) Chimeric pneumococcal

cell wall lytic enzymes reveal important physiological and

evolutionary traits. J Biol Chem 266: 5464–5471.Dideberg O, Charlier P, Dive G, Joris B, Frere JM & Ghuysen

JM (1982) Structure of a Zn2+-containing

D-alanyl-D-alanine-cleaving carboxypeptidase at 2.5 A

resolution. Nature 299: 469–470.Donovan DM, Dong S, Garrett W, Rousseau GM, Moineau S

& Pritchard DG (2006) Peptidoglycan hydrolase fusions



maintain their parental specificities. Appl Environ Microbiol

72: 2988–2996.Dziarski R & Gupta D (2006) The peptidoglycan recognition

proteins (PGRPs). Genome Biol 232: 1–13.Farka�sovska J, Godany A & Vl�cek �C (2003) Identification and

characterization of an endolysin encoded by the Streptomyces

aurefaciens phage l1/6. Folia Microbiol 48: 737–744.Farka�sovska J, Klu�car L’, Vl�cek �C, Kokavec J & Godany A (2007)

Complete genome sequence and analysis of the Streptomyces

aureofaciens phage l1/6. Folia Microbiol 52: 347–358.Finn RD, Tate J, Mistry J et al. (2008) In the Pfam protein

families database. Nucleic Acids Res 36: D281–D288.Fischetti VA (2010) Bacteriophage endolysins: a novel

anti-infective to control Gram-positive pathogens. IJMM

300: 357–362.Foster SJ (1991) Cloning, expression, sequence analysis and

biochemical characterization of an autolytic amidase of

Bacillus subtilis 168 trpC2. J Gen Microbiol 137: 1987–1998.Hermoso JA, Garcia JL & Garcia P (2007) Taking aim on

bacterial pathogens: from phage therapy to enzybiotics. Curr

Opin Microbiol 10: 461–472.Hopwood DA (2007) Streptomyces in Nature andMedicine: The

Antibiotic Makers. Oxford University Press, New York, NY.

Kornd€orfer IP, Danzer J, Schmelcher M, Zimmer M, Skerra A

& Loessner MJ (2006) The crystal structure of the

bacteriophage PSA endolysin reveals a unique fold

responsible for specific recognition of Listeria cell walls.

J Mol Biol 364: 678–689.Kretzer JW, Lehmann R, Schmelcher M, Banz M, Kim KP,

Korn C & Loessner MJ (2007) Use of high-affinity cell

wall-binding domains of bacteriophage endolysins for

immobilization and separation of bacterial cells. Appl

Environ Microbiol 73: 1992–2000.Larkin MA, Blackshields G, Brown NP et al. (2007) ClustalW

and Clustal X version 2.0. BMC Bioinformatics 23: 2947–2948.Loessner MJ (2005) Bacteriophage endolysins – current

state of research and applications. Curr Opin Microbiol 8:

480–487.Loessner MJ, Kramer K, Ebel F & Scherer S (2002) C-terminal

domains of Listeria monocytogenes bacteriophage murein

hydrolases determine specific recognition and high-affinity

binding to bacterial cell wall carbohydrates. Mol Microbiol

44: 335–349.Maleki H & Mashinchian O (2011) Characterization of

Streptomyces isolates with UV, FTIR spectroscopy and HPLC

analyses. Bioimpacts 1: 47–52.

Mao J, Schmelcher M, Harty WJ, Foster-Frey J & Donovan

DM (2013) Chimeric Ply187 endolysin kills Staphylococcus

aureus more effectively than the parental enzyme. FEMS

Microbiol Lett 342: 30–36.Marchler-Bauer A, Zheng C, Chitsaz F et al. (2013) CDD:

conserved domains and protein three-dimensional structure.

Nucleic Acids Res 41: D348–D352.Mayer MJ, Garefalaki V, Spoerl R, Narbad A & Meijers R

(2011) Structure-based modification of a Clostridium

difficile-targeting endolysin affects activity and host range.

J Bacteriol 193: 5477–5486.Rodriguez-Rubio L, Mart�ınez B, Rodr�ıguez A, Donovan DM &

Garcia P (2012) Enhanced staphylolytic activity of the

Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88

HydH5 virion-associated peptidoglycan hydrolase: fusions,

deletions, and synergy with LysH5. Appl Environ Microbiol

78: 2241–2248.Sambrook J & Russel DW (2001) Molecular Cloning: A

Laboratory Manual, 3rd edn. Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, NY.

Sass P & Bierbaum G (2007) Lytic activity of recombinant

bacteriophage phi11 and phi12 endolysins on whole cells

and biofilms of Staphylococcus aureus. Appl Environ

Microbiol 73: 347–352.Schmelcher M, Shabarova T, Eugster MR, Eichenseher F,

Tchang VS, Banz M & Loessner MJ (2010) Rapid multiplex

detection and differentiation of Listeria cells by use of

fluorescent phage endolysin cell wall binding domains. Appl

Environ Microbiol 76: 5745–5756.Schmelcher M, Tchang VS & Loessner MJ (2011) Domain

shuffling and module engineering of Listeria phage

endolysins for enhanced lytic activity and binding affinity.

Microb Biotechnol 4: 651–662.Zhang Y (2008) I-TASSER server for protein 3D structure

prediction. BMC Bioinformatics 9: 40.

Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig. S1. 12% SDS-PAGE analysis of expressed fusion pro-

teins.

Fig. S2. I-TASSER tertiary structure prediction of Lyt

µ1/6 CBD.



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Bacteriophage endolysin Lyt μ1/6: characterization of the C-terminal binding domain