RESEARCH LETTER
Bacteriophage endolysin Lyt μ1/6: characterization of the C-terminal binding domain Lenka Tisa´kova´1, Barbora Vidova´1, Jarmila Farkasovska´1 & Andrej Goda´ny1,2 1
Department 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 Tisakova, Institute of Molecular Biology Slovak Academy of bravska cesta 21, Sciences (IMB SAS), Du 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
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, consisting 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 structure 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.
MICROBIOLOGY LETTERS
Editor: Richard Calendar Keywords actinophage l1/6; Streptomyces aureofaciens; in silico analysis; binding; truncations.
Introduction Endolysins are highly evolved enzymes encoded in bacteriophage 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 single-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 carbohydrate components of the host cell wall peptidoglycan and feature rapid binding kinetics, high affinity, and extraordinary specificity. Fusion of a bacteriophage endolysin CBD with GFP produces a fluorescent, heterologous fusion product that is able to rapidly recognize and bind to
FEMS Microbiol Lett 350 (2014) 199–208
the host cells of a given species or even to individual serovars (Schmelcher et al., 2011). Such protein constructs have several uses, including visualizing the binding efficiency 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 actinophage l1/6 genome has been sequenced (GenBank ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
L. Tis akov a et al.
200
DQ372923) and analyzed (Farkasovska´ 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 (Farkasovska´ 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 construct 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 truncated 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). Further 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 colored in MS-Word to indicate either invariant or conserved 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 l1/6 CBD PG_binding_1 domain are conserved, a PROTEIN PSI-BLAST analysis (Altschul et al., 1997) against only the streptomycete phage and prophage endolysins and ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
other similar proteins was performed, with the CBD sequence of Lyt l1/6 used as a query. To limit the number of sequences to be analyzed, only those from Streptomyces 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 modular structure 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 growth conditions 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 (Farkasovska´ et al., 2003) for preparation of substrate for binding assays. The E. coli strains were grown at 37 °C in Luria–Bertani medium (Sambrook & Russel, 2001), supplemented 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 of plasmids Standard DNA manipulations were performed essentially as described by Sambrook & Russel (2001). The purified genomic DNA of actinophage l1/6 (Farkasovska´ 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) 199–208
Lyt l1/6 cell wall binding domain
201
(a)
(b) Q7Y4H8 D7NW66 K4IB87 K4I2E3 K4HYE4 K4IBM9
214 182 179 180 184 139
YQTTINGLAYGYGAQGDQVTAVGRALVAHGFGSHYQQGPGPNWTDADTENYADYQRSLGYTGQAADGVPGSDSLRQLLGTLPGGRTVSLAHVIAAAQADPPAAQGHQTYGPDVQIVEQA---LADEGLLDQQWVDG-SFGSRTVSAYAAWQRRCGYSGSGADGIPGKASLDRLAAAHGFTTTD -------------------------------------TPKP--------------------------------------------VIDLSKVVTAARTNPPMAKRTVTY-AGVADVKAW---LIAEGLLVKSDTDG-HFGQRVLDAYKAWQRRCGYSGAAADGVPGMTSLRKLAVKHGRTVTA YQVTINGLKYGYGAQGSHVTTVGKALVAKGFGKHYAEGPGPTWSDADTLNYADFQRSLGYSGSDADGVPGEGSLKTLLGSLPGASAPAPAAKPAAKKYEPFPGASFFKRAPKSAIVTAMGKRLVAEGCGVYSSGPGPQWTESDRKSYAKWQRKLGYTGSAADGYPGKASWDKLHVPEV----YQVTINGLKYGYDAYGDHVTKVGQALVAKGHGDHYASGPGPRWTDADTLNYADFQRSLGYSGADADGVPGESSLRALLGYLPGATATV--------KYEPFPGATFFKNAPRSAIVAAMGKRLVAEGCSAYSSGPGPQWTEADRLSYQKWQRKLGYSGADADGWPGKTSWDKLRVPEV----MDSMRDRVDARLD-----------------------DKPKPKPPAPKPTPPAKPKPTPPKKP-----------------------AVSLARLITAARIDPAKKGTPVSYAGARVVEQAL----AAEGLLDRALIDG-HFGTATRTAYGRWQARQGYRGTAADGVPGRASLGALAARHGFTVTA KRARRDPYLYGYG---------------------YPDVAGSLSADPDASRFGYKHASTSKPAVAPKPKPKPKPKP--------------------KAYEPFPGAAFFKREPKSAIVTAMGKRLVAVGCSAYKSGPGPQWTDADRASYAKWQRKRGYSGADADGWPGKASWDALKVPKV----. . :* . * * : :* ** : ** *: *** ** :* * . .
393 278 356 350 315 276
(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.
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 vectors. PCR products were purified using a GeneJet PCR Purification Kit (Thermo Scientific), digested with appropriate restriction enzymes, and introduced to expression vectors pET21a+ or pET21d+ using a Rapid Ligation Kit (Thermo Scientific). The gfp coding sequence from template 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 FEMS Microbiol Lett 350 (2014) 199–208
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 Lyt l1/6 CBD gene products Escherichia coli BL21(DE3) cells harboring the recombinant plasmids (Table 1) were grown overnight in LB ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
GFP-f: (HindIII) CCCAAGCTTATGAGTAAAGGAG AAGAA** GFP-r: (XhoI) CCGCTCGAGTTTGTATAGTTCATCCAT**
PG-f: (NcoI) CATGCCATGGCACCTACGGGCCC GATGTGC** Gp50-r: (HindIII) CCCAAGCTTGTCGGTGGTGGT GAAGCCGTGGGCGGC** 3H-f: (NcoI) CATGCCATGGCACCCGATGTGCA GATCGTG** 3H-r: (HindIII) CCCAAGCTTGGCGGCGGCGAGGCG**
Gp50-f: (NdeI) GGAATTCCATATGCCCGACTTG TGGATGCCTGGTGC** Gp50-r: (HindIII) CCCAAGCTTGTCGGTGGTGGT GAAGCCGTGGGCGGC** CBD-f: (NdeI) GGAATTCATATGCGGTACCAGA CCACCATCAAC** Gp50-r: (HindIII) CCCAAGCTTGTCGGTGGTGGT GAAGCCGTGGGCGGC** BS-f: (NcoI) CATGCCATGGCATACGCCGACTAC CAGCGG** Gp50-r: (HindIII) CCCAAGCTTGTCGGTGGTGGT GAAGCCGTGGGCGGC**
Primers used for the amplification of lyt inserts (with restriction sites included)
*Subcloning of the gene for gfp is underlined. **Restriction sites (in italics) are underlined.
pET21a-gfp
pET21d-lytCBD-3H-gfp*
pET21d-lytCBD-PG-gfp*
pET21d-lytCBD-BS-gfp*
pET21a-lytCBD-gfp*
pET21a-lyt-gfp*
Name
Constructs
715
186
217
389
599
1180
Size of lyt inserts in bp
P324–A386
T329–D393
Y264–D393
GFP
3H-GFP
PG-GFP
BS-GFP
CBD-GFP
V1 – D393
Y214–D393
Name LYT-GFP
Sequence range of lyt inserts
26.5
33.4
34.5
40.7
48.1
69
Size in kDa without His-Tag
Fusion protein products
Truncation of CBD with preserved potential binding site = ‘BS’ Truncation of CBD with whole PG_binding_1 domain = ‘PG’ Truncation of CBD with only three helices of PG_binding_ domain = ‘3H’ Negative control for binding assays = GFP
Function of whole CBD = ‘CBD’
Function of whole Lyt l1/6 = ‘LYT’
Rationale
+++
++
++
++++
Lysis
Binding activity to the streptomycete substrate
Table 1. Fusion constructs and their protein products showing characterization, the oligonucleotides used for PCR amplification of the lyt inserts, the rationale for each construct’s design and the actual binding activity of fusion proteins in binding assay. The intensity of binding to the streptomycete cell walls is marked with + for the weakest fluorescence, which was not observed, ++ for weak fluorescence, +++ for medium fluorescence, ++++ for the strongest ( for no) fluorescence under fluorescence microscopy conditions
202 L. Tis akov a et al.
FEMS Microbiol Lett 350 (2014) 199–208
Lyt l1/6 cell wall binding domain
medium containing ampicillin (100 lg mL 1). The culture 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-bD-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 Filter 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 sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (Supporting Information, Fig. S1). SDS-PAGE was performed on a 12% gel using a Spectra Multicolor Broad Range Ladder (Thermo Scientific) as the molecular size marker. Gels were stained in Coomassie stain for 15 min and then destained via conventional methods. Proteins were stored at 20 °C until assayed. Binding activity assays and fluorescence microscopy 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, resuspended 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 incubated 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 lL of Tris-HCl buffer, and the pellet was resuspended in 50 lL Tris-HCl. Cells were visualized on poly-L-lysinetreated slides using fluorescence microscopy. All images were obtained using a LEICA DM2500 microscope equipped with a LEICA DFC290 HD camera and LAS software.
FEMS Microbiol Lett 350 (2014) 199–208
203
Results and discussion Analysis of Streptomyces phage endolysins and their modular structure
Presently (November 2013), GenBank contains the complete nucleotide sequences of nine Streptomyces bacteriophages, 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 (Accession: JX182370), SV1 (Accession: JX182371), and TG1 (Accession: JX182372). Among them, six putative endolysins 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 peptidoglycan (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 proteins possessing this domain have not yet been characterized, 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 positions (Fig. 1b). Nearly all identical amino acids were found at the protein C-termini, where the binding properties 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
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
YP_006906203.1 NC_018836.1 YP_006906963.1 NC_018848.1
YP_006907195.1 NC_018853.1
S. aureofaciens
S. avermitilis
S. coelicolor A3(2)
S. coelicolor A3(2)
S. venezuelae
S. avermitilis
l1/6
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
ΦSASD1
R4
ΦHau3
SV1
TG1
K4IBM9
K4HYE4
K4I2E3
K4IB87
D7NW66
Q7Y4H8
Uniprot
831
948
1053
1071
837
1182
nt
Sequence length
276
315
350
356
278
393
aa
29 818
33 449
37 152
37 084
29 600
42 132
Mass (Da)
PG_binding_1 = Putative peptidoglycan binding domain; PGRP = Peptidoglycan recognition proteins (PGRPs).
YP_579222.1 NC_007967.1 YP_003714747.1 NC_014229.1 YP_006990140.1 NC_019414.1
RefSeq
Host
Streptomyces Phage
Endolysin ID
15 771–16 601
16 368–17 315
21 373–22 425
21 877–22 947
16 596–17 432
36 463–37 644
Genomic range
cl11438 NLPC_P60; NlpC/P60 family (unknown function)
Amidase_domain. N-acetylmuramoyl-L-alanine amidase activity Amidase_domain. N-acetylmuramoyl-L-alanine amidase activity –
–
–
Catalytic
Conserved domains
COG3409 Putative peptidoglycan-binding domain-containing protein cd06583 PGRP pfam01471 PG_binding_1
cd06583 PGRP
pfam01471 PG_binding_1 pfam01471 PG_binding_1 cd06583 PGRP
Binding
Table 2. The list of putative endolysins from Streptomyces phages found in GenBank (NCBI search – keywords ‘phage endolysin’ and ‘streptomyces’; verified in UniProt). Included features: host (lab host), references, database IDs, genomic range and gene length, protein mass and length, and identified conserved domains; nt – nucleotides; aa – amino acids
204 L. Tis akov a et al.
FEMS Microbiol Lett 350 (2014) 199–208
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extended analysis against the Streptomyces phage and prophage endolysins. Although the alignment showed less overall amino-acid conservation (Fig. 2) than the six identified 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 numbering) were all invariant (Fig. 2). This conservation suggests 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 Streptomyces 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 l1/6 C-terminal domain. Analysis of the endolysin Lyt l1/6 modular structure
Previous bioinformatics analysis suggested that the endolysin Lyt l1/6 is a modular protein composed of two
Q7Y4H8 D7NW66 K4IB87 K4I2E3 K4HYE4 K4IBM9 G8XHL4 F8JK13 I7A9I3 Q9ZX99 L1L7W7 L7ETE1 H0BPX9 I2MXW1 K1UV81 I0CEJ4 D0UZA7 D9W712 D9XZ47 D9W669 E2PZH7 G2P5J6 F3Z9F2 D9UFT6 D6AD83 G0Q9L7 H0B7I0 B1VX31 K4REH3 K4RC28 B1W438 G0PXR4 I2N4B2
324 209 288 282 238 207 491 499 288 294 332 202 234 234 284 242 376 218 379 221 199 222 215 215 138 223 180 223 227 1 240 240 230
205
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 (Farkasovska´ et al., 2003), but no further experiments were carried out to determine its function. The supposed catalytic domain at its N-terminus, 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 secondary 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 streptomycete peptidoglycan are still poorly understood. According to the predicted 3D structure of Lyt l1/6 CBD region by I-TASSER, Y357 and W360 occupied the domain core, and Y357 interacted with the highly
PDVQIVEQALAD--------EGLLDQQ--WVDGSFGSRTVSAYAAWQR-RCG--YSGSGADGIPGKASLDRLAAAHGFTTTD AGVADVKAWLIA--------EGLLVKS--DTDGHFGQRVLDAYKAWQR-RCG--YSGAAADGVPGMTSLRKLAVKHGRTVTA PKSAIVTAMGKR-----LVAEGCGVYSS-GPGPQWTESDRKSYAKWQR-KLG--YTGSAADGYPGKASWDKLHVPEV----PRSAIVAAMGKR-----LVAEGCSAYSS-GPGPQWTEADRLSYQKWQR-KLG--YSGADADGWPGKTSWDKLRVPEV----KKGTPVSYAGARVVEQALAAEGLLDRAL-IDG-HFGTATRTAYGRWQA-RQG--YRGTAADGVPGRASLGALAARHGFTVTA PKSAIVTAMGKR-----LVAVGCSAYKS-GPGPQWTDADRASYAKWQR-KRG--YSGADADGWPGKASWDALKVPKV-----HPDDVFTVELA-----LVDEGLLDREW-A-DGSFGTRTITAYAELQR-RYG--YSGQMADGIPGTESLTRLGRAHGFTVR-HPDDVFTVELA-----LVDEGLLDREW-A-DGSFGTRTITAYAELQR-RYG--YSGQMADGIPGTESLTRLGRAHGFTVRPKSAIVTAMGKR-----LVAEGCGVYSS-GPGPQWTESDRKSYAKWQR-KLG--YTGSAADGYPGKASWDKLHVPEV----PKSPIVTAMGKR-----LVAEGCSAYRS-GPGAQWTNADKASYAKWQR-KRG--YSGADADGWPGKTTWDALKVPKV----TDVRLVEEALAA--------EGLLERG--YVDGSFGTRTIEAYAAWQRSRAGGSYRGRDADGVPGRASLTRLGDRHGFTVIA -FPADVRPVEAA-----LVAEGLLDPTF-GGDGSFGSHTVDAYAAFQR-QQG--FTGANADGIPGESTLAALGSRHGFTVAA ADVKIVEAALQK--------EGLLGASY-AKDGSFGSLTRAAYSAWQR-RCG--YSGSAADGIPGKASLEKLGVKRGFKVKA RPGTPVSYPGVKTVEKALVKEGLLTAGL-ADG-HYGTATKDAYAAWQR-RLG--YTGGAADGIPGQASLKKLATKHGFTVTP ADVKIVEAALKA--------EGLLAATY-AADGSWGTKTDTAYDAFRR-KMG--YTGSAATGSVGLESLKKLAARHGFTAKA RKSPIVTAMGRR-----LVAEGCGRYSQ-GPGPNWTNADKASYAAYQR-KLG--YSGAAADGIPGKTSWDKLRVPKQL---RKSPLITAMGRR-----LVAEGCGKYKQ-GPGPNWTNVDKASYSAWQR-KLG--YSGTAADGIPGKASWDKLRVPKQL---RDSEIVTAMGKR-----LVAEDCDHYQE-GPGPEWTDADQESYAAWQR-KLG--FSGDDADGIPGEVSWDKLRVPQD----AVNDQVTRLGEQ-----LVRKGFGRYYADGPGPRWSEADRRNVEAFQR-AQG--WRGGAADGYPGPETWRRLFLS------RESKIITAMGKR-----LVAEGCDRYEE-GPGPEWTDADKKSYAAWQR-KLG--YSGDDADGIPGKKSWDKLRVPNV----RRSPIVTAMGRR-----LVAEGCGRYEI-GPGPAWSEADRRSYAAWQR-KLG--YSGAAADGIPGKTSWDRLKVPNT----RQSKIITAMGKR-----LVAEGCGRYEE-GPSPEWTEADRKSYAAWQH-KLG--YSGDGADGVPGKASWDKLRVPNV----PKSALVTAMGRR-----LVAEGCSAYAE-GPGPQWTAADRASYAKWQR-KLG--YTGADADGWPGAASWRALKVPGV----PKSALVTAMGRR-----LVAEGCSAYAE-GPGPQWTAADRTSYAKWQR-KLG--YTGPDADGWPGAASWRALKVPGV----RNSAIVTAMGKR-----LVAEGCGRYTV-GPGPAWSEADRKSYAAWQR-KLG--YTGGDADGIPGKSSWDRLKVPNV----RNSAVVTAMGRR-----LVSEGCGRYTV-GPGPAWSEADRKSYAAWQR-KLG--YTGGDADGIPGKSSWDRLKVPNV----RNSAVITAMGKR-----LVSEGCGRYTV-GPGPAWSEADRKSYAAWQR-KLG--YTGGDADGVPGKSSWDRLKVPNV----RNSAVVTAMGRR-----LVSEGCGRYTV-GPGPAWSEADRTSYAAWQR-KLG--YTGGDADGIPGKSSWDRLKVPNV----QKSPVITAMGRR-----LVAEGCGRYEE-GPGPEWTEADRRSYAAWQQ-KQG--FKGKDADGIPGRVTWERLKVPNGPN----MKTVEAALVA--------EDLLSKA--LADGHFG--TATAYAAWQR-RCG--WSIDDADGTPDLASLTELGKRRGFDVKE PSSPVVTAMGRR-----LSAEGCGAYAV-GPGPRWTEADRRSYAAWQR-KLG--FRGAEADGWPGRTSWNALKVPYTTKSPPSSPVVTAMGRR-----LSAEGCGAYAV-GPGPRWTEADRRSYAAWQR-KLG--FRGAEADGWPGRTSWNALKVPYTTKSPPSSPVITAMGRR-----LLAEGCGEYAV-GPGPRWSEADRRSYARWQR-KLG--FRGADADGWPGAASWNALKVPHTP---: . . : : * : * * . : *
393 278 356 350 315 276 561 569 356 362 404 274 304 310 353 311 445 286 446 289 267 290 283 283 206 291 248 291 297 65 313 313 299
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).
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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 conserved 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 frequently 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 recognition of hydrophobic ligands, which can occasionally be associated with peptidoglycans (Betts & Russell, 2003). These postulated roles of the conserved amino-acid residues will require additional experimental verification. In the next part of the present study, the effects of truncations 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 notably between clostridial and pneumococcal peptidoglycan 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
(a)
1
2
3
4
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 characterizing 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 determine 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 production 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 CBDGFP 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 5
6
7
8
(b)
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).
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Lett 350 (2014) 199–208
Lyt l1/6 cell wall binding domain
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 contained 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. aureofaciens 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 visualize 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 disease 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 amplification 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
FEMS Microbiol Lett 350 (2014) 199–208
207
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
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
L. Tis akov a et al.
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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. (2003) Identification and Farkasovska´ J, Goda´ny A & Vlcek C characterization of an endolysin encoded by the Streptomyces aurefaciens phage l1/6. Folia Microbiol 48: 737–744. Kokavec J & Goda´ny A (2007) Farkasovska´ J, Kluca´r L’, Vlcek C, 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 and Medicine: 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.
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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, Martınez B, Rodrıguez 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 proteins. 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 Lenka Tisa´kova´1, Barbora Vidova´1, Jarmila Farkasovska´1 & Andrej Goda´ny1,2 1
Department 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 Tisakova, Institute of Molecular Biology Slovak Academy of bravska cesta 21, Sciences (IMB SAS), Du 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
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, consisting 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 structure 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.
MICROBIOLOGY LETTERS
Editor: Richard Calendar Keywords actinophage l1/6; Streptomyces aureofaciens; in silico analysis; binding; truncations.
Introduction Endolysins are highly evolved enzymes encoded in bacteriophage 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 single-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 carbohydrate components of the host cell wall peptidoglycan and feature rapid binding kinetics, high affinity, and extraordinary specificity. Fusion of a bacteriophage endolysin CBD with GFP produces a fluorescent, heterologous fusion product that is able to rapidly recognize and bind to
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the host cells of a given species or even to individual serovars (Schmelcher et al., 2011). Such protein constructs have several uses, including visualizing the binding efficiency 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 actinophage l1/6 genome has been sequenced (GenBank ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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DQ372923) and analyzed (Farkasovska´ 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 (Farkasovska´ 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 construct 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 truncated 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). Further 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 colored in MS-Word to indicate either invariant or conserved 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 l1/6 CBD PG_binding_1 domain are conserved, a PROTEIN PSI-BLAST analysis (Altschul et al., 1997) against only the streptomycete phage and prophage endolysins and ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
other similar proteins was performed, with the CBD sequence of Lyt l1/6 used as a query. To limit the number of sequences to be analyzed, only those from Streptomyces 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 modular structure 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 growth conditions 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 (Farkasovska´ et al., 2003) for preparation of substrate for binding assays. The E. coli strains were grown at 37 °C in Luria–Bertani medium (Sambrook & Russel, 2001), supplemented 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 of plasmids Standard DNA manipulations were performed essentially as described by Sambrook & Russel (2001). The purified genomic DNA of actinophage l1/6 (Farkasovska´ 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) 199–208
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(a)
(b) Q7Y4H8 D7NW66 K4IB87 K4I2E3 K4HYE4 K4IBM9
214 182 179 180 184 139
YQTTINGLAYGYGAQGDQVTAVGRALVAHGFGSHYQQGPGPNWTDADTENYADYQRSLGYTGQAADGVPGSDSLRQLLGTLPGGRTVSLAHVIAAAQADPPAAQGHQTYGPDVQIVEQA---LADEGLLDQQWVDG-SFGSRTVSAYAAWQRRCGYSGSGADGIPGKASLDRLAAAHGFTTTD -------------------------------------TPKP--------------------------------------------VIDLSKVVTAARTNPPMAKRTVTY-AGVADVKAW---LIAEGLLVKSDTDG-HFGQRVLDAYKAWQRRCGYSGAAADGVPGMTSLRKLAVKHGRTVTA YQVTINGLKYGYGAQGSHVTTVGKALVAKGFGKHYAEGPGPTWSDADTLNYADFQRSLGYSGSDADGVPGEGSLKTLLGSLPGASAPAPAAKPAAKKYEPFPGASFFKRAPKSAIVTAMGKRLVAEGCGVYSSGPGPQWTESDRKSYAKWQRKLGYTGSAADGYPGKASWDKLHVPEV----YQVTINGLKYGYDAYGDHVTKVGQALVAKGHGDHYASGPGPRWTDADTLNYADFQRSLGYSGADADGVPGESSLRALLGYLPGATATV--------KYEPFPGATFFKNAPRSAIVAAMGKRLVAEGCSAYSSGPGPQWTEADRLSYQKWQRKLGYSGADADGWPGKTSWDKLRVPEV----MDSMRDRVDARLD-----------------------DKPKPKPPAPKPTPPAKPKPTPPKKP-----------------------AVSLARLITAARIDPAKKGTPVSYAGARVVEQAL----AAEGLLDRALIDG-HFGTATRTAYGRWQARQGYRGTAADGVPGRASLGALAARHGFTVTA KRARRDPYLYGYG---------------------YPDVAGSLSADPDASRFGYKHASTSKPAVAPKPKPKPKPKP--------------------KAYEPFPGAAFFKREPKSAIVTAMGKRLVAVGCSAYKSGPGPQWTDADRASYAKWQRKRGYSGADADGWPGKASWDALKVPKV----. . :* . * * : :* ** : ** *: *** ** :* * . .
393 278 356 350 315 276
(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.
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 vectors. PCR products were purified using a GeneJet PCR Purification Kit (Thermo Scientific), digested with appropriate restriction enzymes, and introduced to expression vectors pET21a+ or pET21d+ using a Rapid Ligation Kit (Thermo Scientific). The gfp coding sequence from template 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 FEMS Microbiol Lett 350 (2014) 199–208
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 Lyt l1/6 CBD gene products Escherichia coli BL21(DE3) cells harboring the recombinant plasmids (Table 1) were grown overnight in LB ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
GFP-f: (HindIII) CCCAAGCTTATGAGTAAAGGAG AAGAA** GFP-r: (XhoI) CCGCTCGAGTTTGTATAGTTCATCCAT**
PG-f: (NcoI) CATGCCATGGCACCTACGGGCCC GATGTGC** Gp50-r: (HindIII) CCCAAGCTTGTCGGTGGTGGT GAAGCCGTGGGCGGC** 3H-f: (NcoI) CATGCCATGGCACCCGATGTGCA GATCGTG** 3H-r: (HindIII) CCCAAGCTTGGCGGCGGCGAGGCG**
Gp50-f: (NdeI) GGAATTCCATATGCCCGACTTG TGGATGCCTGGTGC** Gp50-r: (HindIII) CCCAAGCTTGTCGGTGGTGGT GAAGCCGTGGGCGGC** CBD-f: (NdeI) GGAATTCATATGCGGTACCAGA CCACCATCAAC** Gp50-r: (HindIII) CCCAAGCTTGTCGGTGGTGGT GAAGCCGTGGGCGGC** BS-f: (NcoI) CATGCCATGGCATACGCCGACTAC CAGCGG** Gp50-r: (HindIII) CCCAAGCTTGTCGGTGGTGGT GAAGCCGTGGGCGGC**
Primers used for the amplification of lyt inserts (with restriction sites included)
*Subcloning of the gene for gfp is underlined. **Restriction sites (in italics) are underlined.
pET21a-gfp
pET21d-lytCBD-3H-gfp*
pET21d-lytCBD-PG-gfp*
pET21d-lytCBD-BS-gfp*
pET21a-lytCBD-gfp*
pET21a-lyt-gfp*
Name
Constructs
715
186
217
389
599
1180
Size of lyt inserts in bp
P324–A386
T329–D393
Y264–D393
GFP
3H-GFP
PG-GFP
BS-GFP
CBD-GFP
V1 – D393
Y214–D393
Name LYT-GFP
Sequence range of lyt inserts
26.5
33.4
34.5
40.7
48.1
69
Size in kDa without His-Tag
Fusion protein products
Truncation of CBD with preserved potential binding site = ‘BS’ Truncation of CBD with whole PG_binding_1 domain = ‘PG’ Truncation of CBD with only three helices of PG_binding_ domain = ‘3H’ Negative control for binding assays = GFP
Function of whole CBD = ‘CBD’
Function of whole Lyt l1/6 = ‘LYT’
Rationale
+++
++
++
++++
Lysis
Binding activity to the streptomycete substrate
Table 1. Fusion constructs and their protein products showing characterization, the oligonucleotides used for PCR amplification of the lyt inserts, the rationale for each construct’s design and the actual binding activity of fusion proteins in binding assay. The intensity of binding to the streptomycete cell walls is marked with + for the weakest fluorescence, which was not observed, ++ for weak fluorescence, +++ for medium fluorescence, ++++ for the strongest ( for no) fluorescence under fluorescence microscopy conditions
202 L. Tis akov a et al.
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medium containing ampicillin (100 lg mL 1). The culture 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-bD-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 Filter 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 sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (Supporting Information, Fig. S1). SDS-PAGE was performed on a 12% gel using a Spectra Multicolor Broad Range Ladder (Thermo Scientific) as the molecular size marker. Gels were stained in Coomassie stain for 15 min and then destained via conventional methods. Proteins were stored at 20 °C until assayed. Binding activity assays and fluorescence microscopy 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, resuspended 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 incubated 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 lL of Tris-HCl buffer, and the pellet was resuspended in 50 lL Tris-HCl. Cells were visualized on poly-L-lysinetreated slides using fluorescence microscopy. All images were obtained using a LEICA DM2500 microscope equipped with a LEICA DFC290 HD camera and LAS software.
FEMS Microbiol Lett 350 (2014) 199–208
203
Results and discussion Analysis of Streptomyces phage endolysins and their modular structure
Presently (November 2013), GenBank contains the complete nucleotide sequences of nine Streptomyces bacteriophages, 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 (Accession: JX182370), SV1 (Accession: JX182371), and TG1 (Accession: JX182372). Among them, six putative endolysins 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 peptidoglycan (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 proteins possessing this domain have not yet been characterized, 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 positions (Fig. 1b). Nearly all identical amino acids were found at the protein C-termini, where the binding properties 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
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
YP_006906203.1 NC_018836.1 YP_006906963.1 NC_018848.1
YP_006907195.1 NC_018853.1
S. aureofaciens
S. avermitilis
S. coelicolor A3(2)
S. coelicolor A3(2)
S. venezuelae
S. avermitilis
l1/6
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
ΦSASD1
R4
ΦHau3
SV1
TG1
K4IBM9
K4HYE4
K4I2E3
K4IB87
D7NW66
Q7Y4H8
Uniprot
831
948
1053
1071
837
1182
nt
Sequence length
276
315
350
356
278
393
aa
29 818
33 449
37 152
37 084
29 600
42 132
Mass (Da)
PG_binding_1 = Putative peptidoglycan binding domain; PGRP = Peptidoglycan recognition proteins (PGRPs).
YP_579222.1 NC_007967.1 YP_003714747.1 NC_014229.1 YP_006990140.1 NC_019414.1
RefSeq
Host
Streptomyces Phage
Endolysin ID
15 771–16 601
16 368–17 315
21 373–22 425
21 877–22 947
16 596–17 432
36 463–37 644
Genomic range
cl11438 NLPC_P60; NlpC/P60 family (unknown function)
Amidase_domain. N-acetylmuramoyl-L-alanine amidase activity Amidase_domain. N-acetylmuramoyl-L-alanine amidase activity –
–
–
Catalytic
Conserved domains
COG3409 Putative peptidoglycan-binding domain-containing protein cd06583 PGRP pfam01471 PG_binding_1
cd06583 PGRP
pfam01471 PG_binding_1 pfam01471 PG_binding_1 cd06583 PGRP
Binding
Table 2. The list of putative endolysins from Streptomyces phages found in GenBank (NCBI search – keywords ‘phage endolysin’ and ‘streptomyces’; verified in UniProt). Included features: host (lab host), references, database IDs, genomic range and gene length, protein mass and length, and identified conserved domains; nt – nucleotides; aa – amino acids
204 L. Tis akov a et al.
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extended analysis against the Streptomyces phage and prophage endolysins. Although the alignment showed less overall amino-acid conservation (Fig. 2) than the six identified 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 numbering) were all invariant (Fig. 2). This conservation suggests 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 Streptomyces 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 l1/6 C-terminal domain. Analysis of the endolysin Lyt l1/6 modular structure
Previous bioinformatics analysis suggested that the endolysin Lyt l1/6 is a modular protein composed of two
Q7Y4H8 D7NW66 K4IB87 K4I2E3 K4HYE4 K4IBM9 G8XHL4 F8JK13 I7A9I3 Q9ZX99 L1L7W7 L7ETE1 H0BPX9 I2MXW1 K1UV81 I0CEJ4 D0UZA7 D9W712 D9XZ47 D9W669 E2PZH7 G2P5J6 F3Z9F2 D9UFT6 D6AD83 G0Q9L7 H0B7I0 B1VX31 K4REH3 K4RC28 B1W438 G0PXR4 I2N4B2
324 209 288 282 238 207 491 499 288 294 332 202 234 234 284 242 376 218 379 221 199 222 215 215 138 223 180 223 227 1 240 240 230
205
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 (Farkasovska´ et al., 2003), but no further experiments were carried out to determine its function. The supposed catalytic domain at its N-terminus, 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 secondary 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 streptomycete peptidoglycan are still poorly understood. According to the predicted 3D structure of Lyt l1/6 CBD region by I-TASSER, Y357 and W360 occupied the domain core, and Y357 interacted with the highly
PDVQIVEQALAD--------EGLLDQQ--WVDGSFGSRTVSAYAAWQR-RCG--YSGSGADGIPGKASLDRLAAAHGFTTTD AGVADVKAWLIA--------EGLLVKS--DTDGHFGQRVLDAYKAWQR-RCG--YSGAAADGVPGMTSLRKLAVKHGRTVTA PKSAIVTAMGKR-----LVAEGCGVYSS-GPGPQWTESDRKSYAKWQR-KLG--YTGSAADGYPGKASWDKLHVPEV----PRSAIVAAMGKR-----LVAEGCSAYSS-GPGPQWTEADRLSYQKWQR-KLG--YSGADADGWPGKTSWDKLRVPEV----KKGTPVSYAGARVVEQALAAEGLLDRAL-IDG-HFGTATRTAYGRWQA-RQG--YRGTAADGVPGRASLGALAARHGFTVTA PKSAIVTAMGKR-----LVAVGCSAYKS-GPGPQWTDADRASYAKWQR-KRG--YSGADADGWPGKASWDALKVPKV-----HPDDVFTVELA-----LVDEGLLDREW-A-DGSFGTRTITAYAELQR-RYG--YSGQMADGIPGTESLTRLGRAHGFTVR-HPDDVFTVELA-----LVDEGLLDREW-A-DGSFGTRTITAYAELQR-RYG--YSGQMADGIPGTESLTRLGRAHGFTVRPKSAIVTAMGKR-----LVAEGCGVYSS-GPGPQWTESDRKSYAKWQR-KLG--YTGSAADGYPGKASWDKLHVPEV----PKSPIVTAMGKR-----LVAEGCSAYRS-GPGAQWTNADKASYAKWQR-KRG--YSGADADGWPGKTTWDALKVPKV----TDVRLVEEALAA--------EGLLERG--YVDGSFGTRTIEAYAAWQRSRAGGSYRGRDADGVPGRASLTRLGDRHGFTVIA -FPADVRPVEAA-----LVAEGLLDPTF-GGDGSFGSHTVDAYAAFQR-QQG--FTGANADGIPGESTLAALGSRHGFTVAA ADVKIVEAALQK--------EGLLGASY-AKDGSFGSLTRAAYSAWQR-RCG--YSGSAADGIPGKASLEKLGVKRGFKVKA RPGTPVSYPGVKTVEKALVKEGLLTAGL-ADG-HYGTATKDAYAAWQR-RLG--YTGGAADGIPGQASLKKLATKHGFTVTP ADVKIVEAALKA--------EGLLAATY-AADGSWGTKTDTAYDAFRR-KMG--YTGSAATGSVGLESLKKLAARHGFTAKA RKSPIVTAMGRR-----LVAEGCGRYSQ-GPGPNWTNADKASYAAYQR-KLG--YSGAAADGIPGKTSWDKLRVPKQL---RKSPLITAMGRR-----LVAEGCGKYKQ-GPGPNWTNVDKASYSAWQR-KLG--YSGTAADGIPGKASWDKLRVPKQL---RDSEIVTAMGKR-----LVAEDCDHYQE-GPGPEWTDADQESYAAWQR-KLG--FSGDDADGIPGEVSWDKLRVPQD----AVNDQVTRLGEQ-----LVRKGFGRYYADGPGPRWSEADRRNVEAFQR-AQG--WRGGAADGYPGPETWRRLFLS------RESKIITAMGKR-----LVAEGCDRYEE-GPGPEWTDADKKSYAAWQR-KLG--YSGDDADGIPGKKSWDKLRVPNV----RRSPIVTAMGRR-----LVAEGCGRYEI-GPGPAWSEADRRSYAAWQR-KLG--YSGAAADGIPGKTSWDRLKVPNT----RQSKIITAMGKR-----LVAEGCGRYEE-GPSPEWTEADRKSYAAWQH-KLG--YSGDGADGVPGKASWDKLRVPNV----PKSALVTAMGRR-----LVAEGCSAYAE-GPGPQWTAADRASYAKWQR-KLG--YTGADADGWPGAASWRALKVPGV----PKSALVTAMGRR-----LVAEGCSAYAE-GPGPQWTAADRTSYAKWQR-KLG--YTGPDADGWPGAASWRALKVPGV----RNSAIVTAMGKR-----LVAEGCGRYTV-GPGPAWSEADRKSYAAWQR-KLG--YTGGDADGIPGKSSWDRLKVPNV----RNSAVVTAMGRR-----LVSEGCGRYTV-GPGPAWSEADRKSYAAWQR-KLG--YTGGDADGIPGKSSWDRLKVPNV----RNSAVITAMGKR-----LVSEGCGRYTV-GPGPAWSEADRKSYAAWQR-KLG--YTGGDADGVPGKSSWDRLKVPNV----RNSAVVTAMGRR-----LVSEGCGRYTV-GPGPAWSEADRTSYAAWQR-KLG--YTGGDADGIPGKSSWDRLKVPNV----QKSPVITAMGRR-----LVAEGCGRYEE-GPGPEWTEADRRSYAAWQQ-KQG--FKGKDADGIPGRVTWERLKVPNGPN----MKTVEAALVA--------EDLLSKA--LADGHFG--TATAYAAWQR-RCG--WSIDDADGTPDLASLTELGKRRGFDVKE PSSPVVTAMGRR-----LSAEGCGAYAV-GPGPRWTEADRRSYAAWQR-KLG--FRGAEADGWPGRTSWNALKVPYTTKSPPSSPVVTAMGRR-----LSAEGCGAYAV-GPGPRWTEADRRSYAAWQR-KLG--FRGAEADGWPGRTSWNALKVPYTTKSPPSSPVITAMGRR-----LLAEGCGEYAV-GPGPRWSEADRRSYARWQR-KLG--FRGADADGWPGAASWNALKVPHTP---: . . : : * : * * . : *
393 278 356 350 315 276 561 569 356 362 404 274 304 310 353 311 445 286 446 289 267 290 283 283 206 291 248 291 297 65 313 313 299
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).
FEMS Microbiol Lett 350 (2014) 199–208
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
L. Tis akov a et al.
206
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 conserved 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 frequently 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 recognition of hydrophobic ligands, which can occasionally be associated with peptidoglycans (Betts & Russell, 2003). These postulated roles of the conserved amino-acid residues will require additional experimental verification. In the next part of the present study, the effects of truncations 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 notably between clostridial and pneumococcal peptidoglycan 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
(a)
1
2
3
4
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 characterizing 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 determine 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 production 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 CBDGFP 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 5
6
7
8
(b)
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).
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Lett 350 (2014) 199–208
Lyt l1/6 cell wall binding domain
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 contained 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. aureofaciens 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 visualize 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 disease 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 amplification 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
FEMS Microbiol Lett 350 (2014) 199–208
207
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
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
L. Tis akov a et al.
208
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. (2003) Identification and Farkasovska´ J, Goda´ny A & Vlcek C characterization of an endolysin encoded by the Streptomyces aurefaciens phage l1/6. Folia Microbiol 48: 737–744. Kokavec J & Goda´ny A (2007) Farkasovska´ J, Kluca´r L’, Vlcek C, 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 and Medicine: 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.
ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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, Martınez B, Rodrıguez 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 proteins. Fig. S2. I-TASSER tertiary structure prediction of Lyt µ1/6 CBD.
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