1 2 3 accepted - applied and environmental...

Insights into Structure and Activity Relationships in the C-1

terminal region of Divercin V41, a Class IIa Bacteriocin with 2

High Antilisterial Activity 3

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Jitka Rihakova1-2

, Vanessa W. Petit3, Katerina Demnerova

2, Hervé Prévost

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Sylvie Rebuffat3 and Djamel Drider

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1UMR INRA SECALIM 1014 ENITIAA-ENVN. Rue de la Géraudière, BP 82225, 44322 Nantes Cedex 7

3, France 2Department of Biochemistry and Microbiology, Institute of Chemical Technology, 166 28 8

Prague 6, Czech Republic, 3Chemistry and Biochemistry of Natural Substances, UMR 5154 CNRS-9

National Museum of Natural History, Department Regulations, Development and Molecular Diversity, 10

Paris, France. 11

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* Corresponding author: Mailing address: ENITIAA. Rue de la géraudière. BP 82225. 44322 Nantes 13

Cedex 3, France. Phone: +33251785542, Fax: +33251785520, E-mail: [email protected] 14

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Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.02266-08 AEM Accepts, published online ahead of print on 30 January 2009

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ABSTRACT 1

Divercin V41 (DvnV41) is a class IIa bacteriocin with potent antilisterial activity isolated from 2

Carnobacterium divergens V41. Previously, we expressed from a synthetic gene, in Escherichia 3

coli Origami, a recombinant DvnV41 named DvnRV41, which possesses four additional amino 4

acids (AMDP) in the N-terminal region that result from enzymatic cleavage, and keeps the 5

initial DvnV41 activity. To unravel the relationships between the structure of DvnRV41 and its 6

particularly elevated activity, we produced by site-directed mutagenesis eight variants in which 7

a single amino acid replacement was specifically introduced in the sequence. The point 8

mutations were designed to change either conserved residues in class IIa bacteriocins, or 9

residues specific to DvnV41 located mainly in the C-terminal region. The fusion proteins were 10

purified from the cytosoluble fractions by immobilized affinity chromatography (IMAC). 11

DvnRV41 and its variants were released from the fusion proteins by enzymatic cleavage, using 12

enterokinase. Purity of DvnRV41 and of the variants was checked by SDS-PAGE, HPLC and 13

mass spectrometry. Antibacterial activity of DvnRV41 and its variants was assessed against 14

different indicator strains including Listeria monocytogenes EGDe and Enterococcus faecalis 15

JH2-2. The activity of all variants appeared to be decreased, as compared to DvnRV41. This 16

drop in activity did not appear to be related to a global conformational change, as determined 17

by circular dichroism. Overall, the variants of DvnRV41 produced in the present study offer 18

interesting insights into structure-activity relationships of class IIa bacteriocins. 19 ACCEPTED


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INTRODUCTION 1

Lactic acid bacteria (LAB) bacteriocins are ribosomally synthesized antimicrobial peptides that 2

could be useful as natural and non-toxic food preservatives. Bacteriocins produced by LAB are known 3

to be active only against Gram-positive bacteria. A few exceptions to this general rule, including 4

bacteriocins from Lactobacillus species (5, 31, 35, 49, 52), AS-48 from Enterococcus faecalis (1), 5

enterocin E50-52 from Enterococcus faecium (50) or thermophylin produced by Streptococcus 6

thermophilus (26), have been described, these bacteriocins exhibiting various degrees of activity 7

against Gram-negative bacteria. 8

A classification of bacteriocins produced by LAB was first proposed by Klaenhammer (30), then 9

modified by Nes et al. (39), and further updated by Cotter et al. (9). Class I and class II bacteriocins are 10

the most abundant and thoroughly studied. Class I bacteriocins, namely lantibiotics, have been widely 11

studied and among them, nisin is routinely used by the food industry as a preservative, due to its high 12

inhibitory activity against a wide range of bacterial pathogens. These bacteriocins are characterized by the 13

presence in their primary structure of post-translationally modified amino acid residues (i.e., lanthionine 14

(Lan) and methylanthionine (MeLan)). Class II of bacteriocins is composed of three subclasses named a, 15

b, c. although additional subclasses d and e are expected. Of these, the most studied subclass is class IIa, 16

also termed pediocin-like bacteriocins (11). 17

Class IIa bacteriocins are anti-listerial, small (< 10 kDa), heat-stable, non-lantibiotic peptides of 18

37 to 48 amino acids containing the consensus sequence YGNGVXCX4C (X denotes any amino acid) in 19

their N-terminal region (11, 13). Class IIa bacteriocins are believed to exert their activity by forming 20

pores in target cell membranes (7), but a recognition step involving the EIItMan

mannose permease of the 21

phosphotransferase system, which would act as a receptor for class IIa bacteriocins (10, 23), would in 22

certain cases take part in the mechanism of action. Studies of the primary structure of class IIa 23

bacteriocins have delineated two distinct domains: a highly conserved hydrophilic and cationic N-24

terminal domain stabilized by a disulfide bond, and an amphiphilic or hydrophobic C-terminal domain, 25

which is less conserved and is stabilized in some peptides, such as pediocin PA-1/AcH, by a second 26

disulfide bond (11). The three-dimensional structure of several one disulfide bond class IIa bacteriocins 27

have been determined (18, 22, 54, 56). Despite high sequence similarity, they do not exhibit a common 28

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structure, but share common elements such as an amphipathic α-helix and most often a β-sheet domain. 1

The N-terminal domain is thought to mediate the initial binding to target cell membranes through 2

electrostatic interactions, while the C-terminal domain would penetrate into the hydrophobic part of the 3

membrane bilayer target cell, causing therefore membrane leakage (16, 36). Class IIa bacteriocins are 4

secreted from their producing cells by dedicated ATP-biding cassettes (ABC transporters) and their 5

accessory proteins, with cleavage of their N-terminal leaders (20). However, some class IIa bacteriocins 6

are secreted by the general secretory pathway (sec) with cleavage of their N-terminal signal peptides (8, 7

29). Class IIa bacteriocins have been the focus of ever increasing level of attention, which has been 8

prompted by their potential use in food protection (6) and in medical area, as anti-viral agents (55), or 9

antibiotic supplements (37). In particular, several structure/activity relationship studies have been 10

undertaken (2, 15, 36, 38, 48, 53, 56). 11

Divercin V41 (DvnV41) is a class IIa bacteriocin, which is naturally produced by 12

Carnobacterium divergens V41, a lactic acid bacterium isolated from fish viscera (40). The broad 13

spectrum of activity of DvnV41 against foodborne pathogenic bacteria, such as L. monocytogenes, in 14

vitro and in vivo (43, 46) has attracted great interest and led to its heterologous production in E. coli (43, 15

59), to enhance peptide yield and simplify its purification procedure (45, 60). DvnV41 is synthesized as a 16

pre-bacteriocin of 66-amino-acids. The 23-residue N-terminal extension is cleaved off to yield the mature 17

43-amino-acid DvnV41 of 4.509 Da, which contains two disulfide bonds formed between Cys10-Cys15 18

(N-terminal disulfide bond conserved in all class IIa bacteriocins) and Cys25-Cys43 (C-terminal disulfide 19

bond specific to the class IIa bacteriocins closely related to DvnV41, such as pediocin PA-1/AcH (13)) 20

(Fig. 1). In addition to the gene encoding the DvnV41 precursor, the genetic system also encodes putative 21

proteins commonly found within bacteriocin operons, including an ATP-dependent transporter, two 22

immunity-like proteins and the two components of a lantibiotic-type signal-transducing system. The

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genetic organization of the nucleotide fragment suggests the occurrence of gene rearrangements, which is 24

consistent with the large repertoire of class IIa bacteriocins (11, 13). 25

Taking advantage of our knowledge of the production of recombinant DvnRV41 in Escherichia 26

coli Origami from a synthetic gene, we here produced eight variants of DvnRV41 designed in order to 27

assess the roles of individual residues, being either common to class IIa bacteriocins, or essentially 28

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specific to those class IIa bacteriocins that include two disulfide bonds. All of them exhibited a lower 1

antibacterial activity than the initial DvnRV41 but in different extents, reflecting their importance for the 2

activity of DvnV41. This genetic approach in combination with circular dichroism (CD) used to assess 3

the conformational modifications induced by the mutations, allowed us to better understanding the role of 4

the selected individual amino acids in the activity of DvnRV41. 5

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MATERIALS AND METHODS 7

8 Bacterial strains, plasmids and bacterial growth conditions. Escherichia coli K-12 strain DHα 9

(Stratagene, Austin, Tex. USA) was used for standard cloning procedures, and E. coli K-12 strain 10

Origami (DE3)(pLysS) (Novagen, Madison, Wis. USA) was used for DvnRV41 expression. E. coli-11

transformants containing the recombinant plasmids (Table 1) were selected on LB agar medium 12

containing ampicillin (100 µg/ml) (Sigma, St. Louis, Mo. USA) 13

Listeria monocytogenes EGDe (19) used as indicator strain was propagated in Elliker broth 14

(Scharlau, Chemie S.A, Spain) and incubated for 24 h at 30°C. Enterococcus faecalis JH2-2 wild-type 15

(59) and its mutant strains resistant to DvnRV41 (4) were grown without shaking in M17 medium 16

(Biokar Diagnostic, Beauvais, France) supplemented with glucose at 0.5 % (w/v) (51). For the growth 17

of Enterococcal mutants, erythromycin (150 µg/ml) or tetracycline (15 µg/ml) was added to the 18

growth medium. 19

Site-directed mutagenesis and synthesis of variants. Point mutations were introduced in dnvRV41 20

through site-directed mutagenesis using the PCR reaction described below. The pCR03 plasmid 21

carrying the synthetic gene dvnRV41 (44) was used as a DNA template. Primers used in the site-22

directed mutagenesis are listed in Table 2. PCR were performed using the Pfu-polymerase (Sigma, 23

Stenheim, Germany). The 50-µl reaction mixture contains 40 ng of DNA template (plasmid 24

pCR03), 0.25 µM of each primer, 0.25 mM of dNTPs and 2.5 U of Pfu-polymerase. After 4 min at 25

95°C, The PCR was initiated at 95°C for 1 min and followed by19 cycles of the following program: 26

denaturation 95°C for 2 min, 55°C for 2 min, 68°C for 10 min and once again 68°C for 15 min. The 27

PCR products were treated for 1 h at 37°C by the restriction enzyme Dpn I (Fermentas, Biogen, 28

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Prague, Czech Republic) to eliminate the methylated DNA template. The size of PCR product was 1

analyzed by 1.5 % agarose gel electrophoresis. Then it was extracted by using QIAquick gel extraction 2

kit (Qiagen) and then introduced in E. coli strain DH5α by heat shock (2 min-42°C). Recombinant 3

strains DH5α harbouring PCR product was grown in LB medium containing ampicillin 100 µg/ml and 4

the novo-synthesized recombinant plasmid was extracted by using the Qiagen miniprep kit 5

(Courtaboeuf, France) according to the manufacturer's recommendations. DNA fragments containing 6

the mutated dvnRV41 genes were obtained by digestion with Nco I and Hind III restriction enzymes 7

and ligated into pET32b vector digested with the same restriction enzymes (Sigma), thus allowing 8

recombinant plasmids (Table 1). Restriction enzymes and T4 DNA ligase were obtained

from New 9

England Biolabs (Beverly, Mass.) and from Promega (Charbonnieres, France), respectively. The 10

cloning strategy used in this work allowed obtaining the translational fusion with thioredoxin TRX-11

(His)6-(Asp)4-Lys-DvnRV41 (DvnRV41-fP). 12

Plasmid pCR03 and recombinant pCR11 to pCR17 (Table 1) were isolated and sequenced to 13

confirm the accuracy of the sequences (Genomic, Czech Republic). Once the sequence was in good 14

agreement with the expected one, the plasmid was introduced into E. coli Origami (DE3)(pLysS) for 15

further analysis. 16

Expression of variants in E. coli Origami. Overnight cultures of E. coli Origami (DE3)(pLysS) 17

harbouring the plasmid pCR11 to 17 were grown aerobically at 37°C in 100 ml of Luria Bertani 18

medium containing ampicillin at 100 µg/ml. When the optical density at 600 nm (measured in a 19

spectrophotometer; Biotek Instruments, Winooski, Vt. USA) had reached 0.6, gene expression was 20

induced by the addition of isopropyl-ß-D-thiogalactopyranoside (IPTG) (Sigma)

to a concentration of 21

1 mM. The cells were grown for additional 3 h, then harvested by centrifugation (15 min, 6 000 x g, 22

4°C) and the cell pellet was kept at –20°C until use for the extraction and purification of the fusion 23

protein. 24

Purification procedures of DvnRV41 and variants. DvnRV41 and its variants were purified as 25

follows: Pellets from 50 ml of a 3-h E. coli/pCR11-17 IPTG-induced cultures were resuspended in 5 26

ml of binding buffer (BB) containing 10 mM imidazole (BB10; pH 7.9) (Amersham Biosciences, 27

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Freiburg, Germany). The cells were disrupted by sonication (Aerosec Industrie, Fecamp, France) in 1

ice-cold water (225 W, five times for 5 min each). The separation of the cytosoluble fractions (CSF) 2

from cytoplasmic insoluble fraction and cell debris was performed by centrifugation (15 000 g, 15 3

min, 4°C). The CSF was incubated at 80°C for 15 min, filtered (0.45 µm-pore-size filter Sartorius, 4

Goettingen, Germany) and then loaded onto a 1 ml nickel His-Trap chelating column (His TrapTM

FF 5

Columns, Amersham Biosciences, Uppsala, Sweden). After loading, the column was eluted with 6

increasing concentrations of imidazole (10 to 500 mM) at pH 7.9. The DvnRV41-fP fusion proteins 7

were found in the 500 mM imidazole elution fraction (EF). This elution process was repeated four 8

times and the different EF obtained were analyzed on sodium dodecyl sulfate-polyacrylamide gel 9

electrophoresis (SDS-PAGE) to localize the fusion proteins. After this first immobilized metal-affinity 10

chromatography (IMAC) purification step, the EF containing the fusion proteins were desalted against

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distilled water by gel filtration (G-25 PD-10 columns, Amersham Biosciences). The DvnRV41-fP 12

fusion proteins were then cleaved with enterokinase

EKMax according to the manufacturer's 13

recommandation (Invitrogen). Separation of DvnRV41 and its variants from TRX-(His)6 -(Asp)4-Lys 14

was achieved by a second IMAC step. 15

Proteins and Immunoblots analysis. Proteins were separated under reducing conditions by 16

Tricine-SDS-PAGE (16.5% polyacrylamide [Sigma]) or glycine-SDS-PAGE (15%

polyacrylamide), 17

which were stained with AgNO3 (Sigma) and

Coomassie blue R-250 (Sigma), respectively. An ultra-18

low-range marker (Sigma) was used as a molecular mass marker (26.6, 17.0, 14.2, 6.5,

3.5, and 1.1 19

kDa). The protein concentration was determined by using the BCA protein assay reagent kit (Pierce, 20

Rockford, IL. USA), with bovine serum albumin as a standard. Immunoblots using the proteins 21

separated by Tricine-SDS-PAGE were achieved with anti-DvnCt-KLH antibodies as previously 22

described (43). 23

The EF containing DvnRV41 or the variants was loaded onto a C8 solid phase extraction cartridge 24

(Sep Pak, Waters), conditioned with acetonitrile (ACN) and equilibrated with 0.1% aqueous formic 25

acid (FA). After sample loading, the cartridge was washed with 0.1% aqueous FA and the elution was 26

performed stepwise with 20%, 30%, 40% and 50% ACN in 0.1 % aqueous FA. The 30% ACN 27

fraction was dried under vacuum, reconstituted in a mixture of ACN and 0.1% FA (50:50, v/v). The 28

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purity of DvnRV41 variants was analyzed by reversed-phase high-performance liquid chromatography 1

(RP-HPLC) using a C18 column (Capcell 5 µm, 4.6 x 250 mm, Interchim, France). Peptides were 2

detected at 226 nm and the peptide fractions were collected manually. Molecular masses and the purity 3

of the peptides were analyzed by matrix-assisted laser desorption ionization time-of-flight mass 4

spectrometry (MALDI-TOF-MS) (Voyager-De-Pro, Applied Biosystems), with α-cyano-4-5

hydroxycinnamic acid as a matrix. The spectra were acquired in linear mode. Electrospray-ionization 6

mass spectrometry (ESI-MS) analyses were carried out on a Qq-TOF/MS instrument (Q-Star Pulsar, 7

Applied Biosystems) to check the presence of the 2 expected disulfide bonds for DvnRV41 and the 8

variants. The concentration of bacteriocins was determined by UV absorption measurement at 280 nm, 9

which was converted to protein concentration using the molecular extinction coefficients calculated 10

from the contribution of individual amino acid residues. 11

Assessment of antilisterial activity. Antibacterial activity of DvnRV41-fP, purified DvnRV41 12

and their variants were assayed by spot-on-law inhibition tests against the sensible L. monocytogenes 13

EGDe strain (Scharlau, Chemie S.A, Spain) and expressed in arbitrary activity units (AU) (40). We 14

used this strain because of its virulence character and whole sequence availability. Ten microliters of 15

each peptide were added to Elliker soft agar plates containing 107 CFU/ml of indicator strain. Plates 16

were incubated for 16 h at 30°C and then inspected by observing the formation of inhibition zones. 17

The diameter of inhibition zone was measured and integrated in the calculation of total activity (Table 18

3). 19

Minimal inhibitory concentration (MIC) determination. Two serial dilutions of DvnRV41 or 20

its variants in Elliker broth were placed into wells of a microtiter plate (Nunc). Each well was 21

inoculated with 50 µl of indicator strain at 105 CFU/ml. The microtiter plate cultures were incubated at 22

30°C for 18 h; the OD600nm was measured at regular intervals of time (1 h) using an UltraMicroplate 23

Reader (Bio-Tek Instruments). MICs were calculated from the highest dilution showing complete 24

inhibition of the tested strain. MIC determination was repeated independently at least three times 25

(Table 4). 26

CD spectroscopy. CD spectra were recorded on a Jasco J-810 instrument equipped with a Peltier 27

effect-based thermostated cell holder. Samples were studied in quartz cells with path lengths of 0.5 28

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mm at 25°C. They were prepared by dissolving the peptides to the concentration of 50 µM either in 1

2,2,2-trifluoroethanol (TFE), TFE/H2O (80:20, v/v) or in 10 mM sodium phosphate (pH 7), in the 2

absence or presence of 50 mM dodecylphosphocholine (DPC) micelles. The spectral contribution from 3

the background was subtracted and the CD signals were normalized to the peptide concentration and 4

expressed as the mean residue weight ellipticity values [θ] in deg cm2 dmol

-1. Samples were scanned 5

over the 190-260 nm wavelength range by recording values every 1 nm with 50 nm min-1

scan rate, 1 s 6

response time and 1 nm band width. Each spectrum was the average of 5 scans. Percentages of the 7

various secondary structures were calculated using the CD spectra deconvolution software Dichroweb 8

(58) (http://www.cryst.bbk.ac.uk/cdweb/html/home.html). An estimate of the different structures 9

theoretically present in DvnRV41 and its variants was calculated using the Psipred programme (28, 10

34; http://bioinf.cs.ucl.ac.uk/psipred/). 11

RESULTS 12

Purification and characterization of DvnRV41 and variants. Eight amino acids were targeted for 13

change in the DvnRV41 variants: Trp19, Gln21, Ala22, Ser23, Val30, Leu35, Ala38, Pro40. They 14

were selected because (i) they are common to class IIa bacteriocins (Trp19, Ala22), or (ii) they are 15

essentially specific of the group of class IIa bacteriocins closely related to DvnV41, which possess two 16

disulfide bonds (Fig.1). They were chosen located mainly in the joining part between the N- and C-17

terminal domains and in the C-terminal part itself. In order to maintain a similar local polarity, each 18

selected amino acid was replaced by another one of a similar polarity group, in agreement with the 19

amino acid Venn diagram (51) leading to variants W19F, Q21S, A22G, S23Y, V30F, L35F, A38V 20

and P40V. The eight selected mutations were created by site-directed mutagenesis in vitro, leading to 21

eight DvnRV41 variants. Each mutation (1 DNA triplet) was specifically introduced into the dvnRV41 22

gene, leading to the substitution of a single amino acid in the DvnRV41 sequence. Expression of the 23

DvnRV41 fusion protein (DvnRV41-fP) and of its variants by the various E. coli Origami hosts 24

harbouring the recombinant plasmids listed in Table 1 was induced by addition of IPTG, as described 25

in Materials and Methods. Induction of the T7 RNA polymerase promoter resulted in the expression

of 26

neo-synthesized polypeptides, which molecular masses were in agreement

with the calculated 27

theoretical masses (data not shown). DvnRV41 and the variants were released enzymatically from the 28

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corresponding fusion proteins. The purification procedure allowed obtaining pure peptides, as depicted 1

in Fig. 2 for variant W19F. Prior to further experiments, the presence of the 2 expected disulfide bonds 2

and the substitutions generated in the DvnRV41 sequence were verified by ESI-MS and MALDI-3

TOF-MS (data not shown). 4

Determination of antibacterial activities. The antibacterial activities of IMAC-immobilized 5

eluted fractions (DvnRV41-fP and its variants) and of enterokinase cleavage products were determined 6

by the agar diffusion assay, as previously reported (40, 43). As shown in Table 3, five fusion proteins 7

were unable to inhibit the growth of L. monocytogenes EGDe. Remarkably, DvnRV41-fP-S23Y 8

exhibited a strong antilisterial activity, similar to that of DvnRV41-fP. The remaining fusion proteins, 9

DvnRV41-fP-A22G and DvnRV41-fP-P40V, both exhibited low activity under the conditions used. 10

It was apparent, however, that following enterokinase cleavage and purification, DvnRV41 and its 11

variants all displayed antilisterial activity (Table 3). As observed with both L. monocytogenes EGDe 12

(19), and Ent. faecalis JH2-2 (4) used as sensitive/indicator strains, MIC measurements (Table 4) 13

showed that variant S23Y retained the highest activity, similar to that of unmodified DvnRV41, while 14

variants W19F and Q21S were the most affected by the amino acid replacements. Finally, the other 15

variants exhibited an intermediate comportment, with a moderately reduced activity in the following 16

order of potency: L35F > V30F=A38V > P40V > A22G > W19F=Q21S. Overall, upon replacements 17

of the selected amino acids, the DvnRV41 MICs were increased by a factor 10 to 200, and 5 to 30 in 18

L. monocytogenes EGDe and Ent. faecalis JH2-2, respectively. 19

The activity of DvnRV41 and its variants was also examined against three Ent. faecalis JH2-2 20

mutants, (∆rpoN, ∆pde and ∆glpQ) (Table 4), recently isolated and shown to exhibit intermediate 21

resistance to DvnRV41 (4). All three Ent. faecalis mutants exhibited higher resistance to DvnRV41 22

than the wild type strain, with MIC values that have increased by a factor 5 (∆pde), 3 (∆glpQ) or 50 23

(∆rpoN). While the wild-type Ent. faecalis JH2-2 strain was sensible to the DvnRV41 variants, the 24

resistant mutants showed a considerable level of resistance to the variants, MICs being enhanced to 25

values ≥ 100 µg/ml for the variants W19F and Q21S (Table 4). The order of efficiency previously 26

determined for the DvnRV41 variants with the wild type strain did not differ with the three Ent. 27

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faecalis JH2-2 mutants resistant to DvnRV41. Of the mutants tested, it was notable that ∆rpoN was 1

least affected by the amino acid replacements, with MICs of the variants being increased by a factor 2 2

to 4 only, while MICs increased by factors 4 to 40 and 2 to 60 were observed for ∆pde and ∆glpQ 3

mutants, respectively . 4

Circular Dichroism (CD) analysis of DvnRV41 and its variants. Since they exhibited the 5

highest loss of antibacterial activity, the variants W19F, Q21S, A22G and P40V were chosen to 6

examine by CD spectroscopy the impact of these mutations on the global conformation of DvnRV41. 7

Their purity and molecular masses were checked by HPLC and MALDI-TOF MS (Supplementary 8

material). CD measurements were performed either in phosphate buffer at pH 7, or in the presence of 9

DPC micelles and in 80% TFE to mimic membrane environments. The CD spectrum of DvnRV41 in 10

aqueous buffer displayed a negative band at 200 nm and a positive band at 230 nm characteristic of 11

random coil conformation. Upon addition of DPC or in the presence of 80 % TFE, the spectrum 12

showed both a β-sheet contribution, as indicated by the minimum around 215 nm, and the 13

characteristics typical of a right-handed helical structure, with negative bands at 207 and 220 nm 14

accompanied by a positive band around 190 nm. Thus, DvnRV41 appeared to be unstructured in 15

aqueous condition and to acquire a mixed α-helix/β-sheet structure in the presence of DPC micelles or 16

in 80% TFE. None of the amino acid replacement appeared to result in a complete loss or 17

modification of the structure. The variants exhibited a very similar behaviour, with a mainly random 18

coil structure in aqueous medium and a mixed α-helix/β-sheet structure in membrane mimicking 19

media, except the W19F variant which differed in that its CD spectrum was similar in all of the 20

conditions used. An estimate of the different secondary structure contribution using the Dichroweb 21

programme (http://www.cryst.bbk.ac.uk/cdweb/html/home.html) (Table 5) allowed a comparison of 22

the global fold of the variants. A slight but significant decrease in the helical content, accompanied by 23

a slight increase in β-sheet content, was observed for all of them and particularly for A22G, both in 24

DPC and TFE environments. A prediction of the DvnV41 three-dimensional structure using the 25

programme Psipred (http://bioinf.cs.ucl.ac.uk/psipred/) afforded it is potentially organised into 11% 26

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helix, 50 % β-sheet and 39% random coil structures, which does not contradict our CD data. The same 1

prediction applied to the variants did not point major changes in the structure either. 2

3 DISCUSSION 4

The content of amino acids stands as important for the folding and bioactivity of bacteriocins. 5

Recently, more than 123 bacteriocins produced by Gram positive and Gram negative bacteria have 6

been classified on the basis of their primary structure and a database has been constituted (21). This 7

original database has revealed that 93.5% of bacteriocins contain at least one glycine, 25 % do not 8

contain cysteine, 32% contain only one pair of cysteines, and the proline content is surprisingly low. 9

Until the industrial use of class IIa bacteriocins becomes authorized, it is important to unravel their 10

mode of action as well as structure and function relationships, in order to be able to optimize their use 11

as food additives or anti-infectives. Clearly, the presence of two cysteine residues evolved in disulfide 12

bond appears as a prerequisite for the antibacterial activity of class IIa bacteriocins (11). However, 13

those bacteriocins which contain two disulfide bonds exhibit a much higher activity as compared to 14

their one disulfide bond counterparts (12, 43); this was hypothesized to be due to a better structuration 15

of the C-terminal domain. Indeed the C-terminal domain and the second disulfide bridge located in 16

this region have been proposed as important for determining the target cell specificity of class IIa 17

bacteriocins (16, 17). 18

Convincingly, DvnV41 has a strong antilisterial activity, making it a potential agent in the fight 19

against the foodborne pathogen L. monocytogenes, which continues to cause major illnesses and 20

industrial damages over the globe. Pediocin PA-1/AcH may be considered as an archetype of the 21

subclass of class IIa bacteriocins that contain two disulfide bonds and have the highest activity (11). 22

Its antibacterial activity would be related to a perturbation of membrane bilayers and chemical 23

modifications of specific amino acids have been previously undertaken to delineate this process (2). A 24

recognition step involving components of the mannose permease is also very probably involved in its 25

mechanism of action (23). Taking into account the previous structure/activity relationship studies for 26

class IIa bacteriocins (2, 15, 36, 38, 48, 53, 56) and the study from Marion and coll. (2) who 27

specifically examined the role of the N-terminal region and of the two disulfide bonds for the 28

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bacteriocin activity, we designed eight variants in order to gain more insights into the structure and 1

activity relationships of DvnV41. As the roles of the disulfide bonds, of the consensus sequence 2

YGNGVXCX4C and of the positively charged residues mainly located in the N-terminal domain had 3

been already studied, we here designed variants containing single amino acid replacements located in 4

the central and the C-terminal parts, using a genetic approach which has proved efficient previously 5

(47). We also took advantage of our knowledge of the production of DvnRV41, a recombinant form of 6

this two-disulfide bond class IIa bacteriocin, which proved keeping the antibacterial activity of the 7

natural bacteriocin (44, 60), in order to develop an efficient production of the desired variants. In order 8

to evidence the precise role of the amino acid side chains, each selected amino acid was replaced by 9

another one belonging to a similar group of polarity, in agreement with the amino acid Venn diagram 10

(51). To investigate the roles of individual amino acids being either common to class IIa bacteriocins 11

(Trp19, Ala22), or essentially specific to two disulfide bond class IIa bacteriocins, we thus compared 12

the antibacterial activities of variants W19F, Q21S, A22G, S23Y, V30F, L35F, A38V and P40V of 13

DvnRV41 and studied the global conformational features of the most affected. 14

A CD analysis of the secondary structure elements of DvnRV41 and its variants in phosphate 15

buffer at pH 7, in 80% TFE (3) and in DPC micelles at pH 7, showed that DvnRV41 and its variants 16

are mainly in an unordered structure in an aqueous environment, while they adopt a mixed 17

conformation containing both regions organized in β-sheet and α-helix in an anisotropic solvent or in 18

the presence of DPC micelles, as previously observed for other class IIa bacteriocins (14, 18, 56, 57). 19

Such a mixed α/β conformation has been also determined by NMR and molecular modelling for other 20

class IIa bacteriocins, curvacin A (22) leucocin A (18) and sakacin P (54), while carnobacteriocin B2 21

is mainly organized in an α-helix (56). The CD data and calculated percentages of different structures 22

(Table 5) showed that the global conformation is not substantially modified by the single amino acid 23

replacements, indicating that no major perturbation of the conformation has occurred upon the amino 24

acid replacements and that the local side chain modification is responsible by itself of the decrease of 25

activity observed for the variants. Even if the three-dimensional structures available for one disulfide 26

bond class IIa bacteriocins (18, 22, 54, 56) exhibit some differences between them, which are 27

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presumably due to the peptides themselves but also to different environments, they are mainly 1

apportioned into mixed α-helix/β-sheet structures, the helix being located at the centre and/or in the C-2

terminal region. However, the solution structure of those class IIa bacteriocins that contain two 3

disulfide bonds has never been determined until now. At the very most, a scheme of the pediocin PA-4

1/AcH structure, resulting from CD measurements in micelle and liposome environments, has been 5

proposed (57). This structure, which is in agreement with the structure predictions arising from the 6

pediocin PA1/AcH amino acid sequence, proposes that the second disulfide bond linking the two 7

cysteines located at the central and very C-terminal parts would fold the C-terminal domain back on 8

the central helix. In the case of DvnRV41, both structure prediction and estimation of the percentages 9

of secondary structure by CD spectra deconvolution do not contradict this hypothesis and indicate that 10

the bacteriocin would include an N-terminal β-sheet domain, as also observed for one disulfide bond 11

bacteriocins, and a short helix located at the centre of the peptide. The activity of our variants may be 12

analysed in the light of this hypothetic structure. 13

As compared to DvnRV41, the activities against L. monocytogenes and Ent. faecium were altered 14

for all variants, except S23Y. All the selected amino acids, but Ser23, thus appeared to be important 15

for the bacteriocin activity. Notably, class IIa bacteriocins contain 1 to 4 tryptophan residues, one of 16

them being highly conserved (11). The three Trp in DvnV41 have been shown previously to be crucial 17

for the activity, since modification of any one of them resulted in an inactivation of the bacteriocin 18

activity (2). An essential role of this residue was also mentioned for sakacin P (15), mesentericin Y105 19

(38) and pediocin PA-1/AcH (36), which is structurally closer to DvnV41. In this study, the 20

replacement of Trp19 in DvnRV41 resulted, as expected, in a deleterious effect on the antibacterial 21

activity, thus confirming its major role in the mechanism of action of DvnV41. It is worth noting that 22

even another aromatic residue such as phenylalanine, chosen to replace Trp in the W19F variant, is not 23

able to maintain the activity. The irreplaceable role of tryptophan in the DvnV41 activity is 24

presumably related to its determinant role in the adsorption and orientation of proteins in membranes, 25

due to its capacity to form hydrogen and hydrophobic bonds with the polar and non-polar groups of 26

polar lipids (24, 25). Other aromatic residues, have been shown to play also an important part in the 27

antibacterial activity of class IIa bacteriocins (41). Indeed, the substitution of Phe33 with serine 28

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abolished the antibacterial activity of carnobacteriocin B2 (42). In our case, introducing a 1

supplementary phenylalanine at position 30 or 35 (variants V30F and L35F) did not increase the 2

activity. DvnV41 has three tyrosine residues and modification by nitration of each of them keeps the 3

activity, while modification of all of them causes a loss of the antilisterial activity (2). In this study, we 4

added a supplementary Tyr residue in the DvnRV41 sequence through the Ser 23 for Tyr substitution, 5

but the resulting variant kept the initial activity of DvnRV41 (Table 4). Thus, introducing a 6

supplementary Tyr in the DvnV41 sequence, at least at position 23 located in the putative helix 7

domain, is not beneficial to the activity. 8

The residue modified in variant Q21S results in a drastic decrease of activity (by a factor 200 on L. 9

monocytogenes). Shortening the side chain and changing an amide function for a hydroxyle one in the 10

putative helix thus result in a dramatic effect on the activity. Variant A22G is also much affected by 11

the amino acid replacement (decrease of activity by a factor 100 on L. monocytogenes). The putatively 12

helical central region thus appears to play a pivotal role for the activity. It is to point out that variant 13

P40V also shows a significant decrease of antibacterial activity (by a factor 20). The importance of 14

proline in the structure and biological properties of peptides and proteins is extensively documented. 15

This particular residue is considered as helix- and β-sheet-breaker (32), due to the lack of possibility of 16

this amino acid to establish a stabilizing hydrogen bond and its subsequent ability to induce bends or 17

hinges in helices. Studies on the structure of bacteriorhodopsin have revealed its major role in 18

transmembrane α-helices (33). In diphteria toxin, proline 345 located in the TH8 helix undergoes a 19

conformational change, which is a prerequisite for the toxin translocation (27). Introduction of proline 20

in the mesentericin Y105 sequence altered significantly the activity of this pediocin-like bacteriocin 21

(38) through perturbation of the helix. It is clear from our CD data that the decrease in activity of 22

variant P40V, observed on both L. monocytogenes and Ent. faecalis, was not related to a profound and 23

global conformational change. This is not really surprising, since in our case the proline is located at 24

the very C-terminus of the sequence. As the C-terminus of class IIa bacteriocins has been proposed to 25

be essential for the activity and the target specificity, the presence of a proline at this position would 26

be hypothesized as more important for recognition by the mannose permease EIItMan

, which is 27

supposed to act as a receptor for class IIa bacteriocins (23), than for interaction with membrane 28

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bilayers. Position 22 appears also critical, since the activity of variant A22G is among the most 1

affected. Taken together these data clearly show that (i) the central region 19-23, which is part of the 2

putative helix and (iii) the very C-terminus are pivotal for the activity, as evidenced by variants W19F, 3

Q21S, A22G, S23Y and P40V, while region 30-38, exemplified by variants V30F, L35F, A38V, 4

appears as less important. It could thus be hypothesized that the central region is responsible for the 5

correct spatial positioning of DvnV41 side chains, required for its interaction with membrane bilayers 6

or/and recognition by the receptor. 7

Resistance of Ent. faecalis to DvnV41 is associated to rpoN, glpQ and pde genes, which encode 8

the σ54

factor that would be involved in the expression of the docking molecule that participates to the 9

recognition mechanism, a putative glycerophosphoryl diester phosphodiesterase (GlpQ) that would 10

participate in degradation of phospholipids and fatty acids, and a protein with a putative 11

phosphodiesterase function (PDE), respectively (4). As compared to the DvnRV41 activity, the 12

activities of its different variants on the Ent. faecalis intermediate resistant strains (∆glpQ and ∆pde) 13

are globally similarly or less affected than those measured on the wild type strain. The highest 14

resistance, which is conferred by rpoN, is associated with a reduced sensitivity to the amino acid 15

replacements, as the MICs are only decreased by a factor 2-4 for ∆rpoN, compared to factors 4-60 for 16

∆pde, and ∆glpQ mutants. 17

Convincingly, the present study underlines the important and different roles of individual amino 18

acids located in the centre of the DvnV41 sequence, such as Trp19, Gln21, or Ser23 and at the C-19

terminus in the antibacterial activity of DvnV41. Indeed, circular dichroism used to examine the effect 20

of single substitutions on the global structure has not revealed any major differences at the structural 21

level. Furthermore, none of the single amino acid replacement is able to increase the activity of the 22

bacteriocin or to change the specificity of the bacteria targeted by the bacteriocin. This study paves the 23

way for further detailed understanding of DvnV41 mechanism of action, using randomized 24

mutagenesis for the production of a great number of variants and determination of the three-25

dimensional structure, which will be our next focus. 26

27

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ACKNOWLEDGEMENTS 1

The authors would like to thank Dr. Paul Cotter for critical reading of the manuscript and English 2

improvement. Research at ENITIAA was partly supported by la Région des Pays de la Loire through 3

VANAM II program. Jitka Rihakova is a recipient of PhD scholarship awarded by French 4

Government. 5

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59. Yagi, Y. and D.B. Clewell. 1980. Recombination-deficient mutant of Streptococcus faecal. J. 19

Bacteriol. 143:966-970 20

60. Yildirim, S, D. Konrad, S. Calvez, D. Drider, H. Prévost, and C. Lacroix. 2007. 21

Production of recombinant bacteriocin divercin V41 by high cell density Escherichia coli 22

batch and fed-batch cultures. Appl. Microbiol. Biotechnol. 77:525-531. 23

24

25

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1 2 3 4 5

TABLE 1: Strains and plasmids used in this study 6 7 Bacterial strain or plasmid Characteristics Reference or

source

Strains

E. coli DH5α recA endA1 gyr A96 thi-1 hsdR17 supE44 ∆lac

U169(Φ80 dlacZ∆M15) deoR F- λ

-

Invitrogen

E. coli Origami ∆ (ara–leu)7697 ∆lacX74 ∆phoA PvuII phoR araD139

ahpC galE galK rpsL F'[lac+ lacI q pro]. Novagen

L. innocua F Indicator organism DSVb

L. monocytogenes EGDe Indicator organism 19

Ent. faecalis JH2-2 Indicator organism 59

Ent. faecalis 35A1 Ent. faecalis JH2-2 (∆ pde), indicator organism 4

Ent. faecalis 36H4 Ent. faecalis JH2-2 (∆ glpQ), indicator organism 4

Ent. faecalis 35H1 Ent. faecalis JH2-2 (∆ rpoN), indicator organism 4

Plasmids

pCR03 DvnRV41, Ampr 44

pCR04 DvnRV41, Cmr,Amp

r 44

pET32b Expression vector, Novagen

pCR11 substitution of W19F, Ampr This work

pCR12 substitution of Q21S , Ampr "

pCR12 substitution of A22G, Ampr "

pCR13 substitution of S23Y, Ampr "

pCR14 substitution of V30F, Ampr "

pCR15 substitution of L35F, Ampr "

pCR16 substitution of A38V, Ampr "

pCR17 substitution of P40V, Ampr "

8

Ampr: ampicillin resistance; Cm

r: chloramphenicol resistance, DSV: Direction des Services 9

Vétérinaires de Nantes, France. 10 11 12 13 14 15 16 17 18 19 20 21

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5

6

7 TABLE 2: Primers used in this study for site directed mutagenesis 8

9 Primer Sequence

W19F – forward 5’-GC TGG GTG GAT TTT GGC CAG GCG AGC GGC-3’

W19F – reverse 5’-GCC GCT CGC CTG GCC AAA ATC CAC CCA GC-3’

Q21S – forward 5’-GCTGGGTGGATTGGGGCTCGGCGAGCGGC-3’

Q21S – reverse 5’-GCCGCTCGCCGAGCCCCAATCCACCCAGC-3’

A22G – forward 5’-GGATTGGGGCCAGGGCAGCGGCTGCATTGGC-3’

A22G – reverse 5’-GCCAATGCAGCCGCTGCCCTGGCCCCAATCC-3’

S23Y – forward 5’-GGCCAGGCGTACGGCTGCATTGGCCAGACC-3’

S23Y – reverse 5’-GGTCTGGCCAATGCAGCCGTACGCCTGGCC-3’

V30F – forward 5’-GGC TGC ATT GGC CAG ACC AAG GTT GGC GGT TGG CTG GGC-3’

V30F – reverse 5’-GCC CAG CCA ACC GCC AAC CCT GGT CTG GCC AAT GCA GCC-3’

L35F – forward 5’-C GTG GTT GGC GGT TGG TTT GGC GGT GCG ATT CCG GGC-3’

L35F – reverse 5’-GCC CGG AAT CGC ACC GCC AAA CCA ACC GCC AAC CAC G-3’

A38V – forward 5’-GGC GGT TGG CTG GGC GGT GTG ATT CCG GGC AAA TGC-3’

A38V – reverse 5’-CGA TTT GCC CGG AAT CAC ACC GCC CAG CCA ACC GCC-3’

P40V – forward 5’-GG CTG GGT GCG ATT GTG GGC AAA TGC-3’

P40V – reverse 5’-GCA TTT GCC CAC AAT CGC ACC CAG CC-3’

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TABLE 3: Total and specific antimicrobial activities of DvnRV41-fP, DvnRV41 and their variants

against L. monocytogenes EGDe

Peptides

Activity

of fusion protein

AUa

Total Activity

of purified peptides

AU

Specific Activity

of purified peptides

AU/µg protein

DvnRV41 3,200 76,800 958.5 W19F 0 400 3.2 Q21S 0 400 4.5 A22G 200 2,400 17.4 S23Y 3,200 51,200 312.6 V30F 0 3,200 24.5 L35F 0 12,800 93.4 A38V 0 6,400 53.8 P40V 200 8,000 65.7

aAntibacterial activities were expressed in arbitrary activity units (AU)

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TABLE 4: Determination of minimal inhibitory concentrations (MICs)a of divercin RV41 variants following enterokinase cleavage and further purification

a

The MICs, expressed in µg/ml, were calculated from the highest dilution showing complete inhibition of the tested strain (OD600nm equals OD600nm of the

blank) and were determined (measured) after 16 h of incubation at 30°C

Peptides

L. monocytogenes

EGDe

Ent. faecalis

JH2-2

Ent. faecalis

(∆pde)

Ent. faecalis

(∆glpQ)

Ent. faecalis

(∆rpoN)

DvnRV41 0.1 ± 0.01 0.6 ± 0.01 3.3 ± 1.11 2.1 ± 1.94 30.0 ± 10.01

W19F 21.1 ± 0.9 21.0 ± 10.5 128.0 ± 0.01 128.0 ± 0.01 128.0 ± 0.01

Q21S 18.3 ± 0.9 21.8 ± 0.01 116.0 ± 0.01 116.0 ± 0.01 116.0 ± 0.01

A22G 8.6 ± 0.01 17.2 ± 8.6 51.8 ± 17.3 21.8 ± 0.01 136.0± 0.01

S23Y 0.1 ± 0.01 1.0 ± 0.2 3.6 ± 0.01 5.5 ± 4.27 40.7 ± 0.01

V30F 1.3 ± 0.4 8.1 ± 0.01 12.2 ± 4.1 8.1 ± 8.13 65.0 ± 0.01

L35F 0.7 ± 0.2 3.2 ± 0.71 8.6 ± 0.01 4.3 ± 2.14 68.5 ± 0.01

A38V 1.5 ± 0.4 7.6 ± 0.01 11.4 ± 3.8 15.1 ± 0.01 60.5 ± 0.01

P40V 2.5 ± 0.9 12.6 ± 2.6 35.9 ± 17.1 38.4 ± 0.01 92.2 ± 30.7

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TABLE 5: CD analysis of the Secondary Structure of DvnRV41 and its variants in (A) phosphate buffer, (B) DPC micelles and (C) 80% TFE, using the CD

spectra deconvolution software Dichroweb.

Peptides α-helix β-sheet Unordered

DvnRV41 2 34 64

W19F 7 32 61

Q21S 5 27 68

A22G 3 34 63

P40V 4 36 60


DvnRV41 15 24 61

W19F 11 27 62

Q21S 7 31 62

A22G 5 31 64

P40V 8 29 63


DvnRV41 14 28 58

W19F 9 31 60

Q21S 6 32 62

A22G 4 34 62

P40V 6 36 58

A

B

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Legends to the figures

FIG 1: (A) Sequence of DvnRV41 fusion protein. (B) Overview of the mutations created in the

primary structure of DvnRV41 by site-directed mutagenesis. Cystein residues are underlined.

Compared to natural DvnV41, the recombinant DvnRV41 contains 4 additional amino acids at the N-

terminus (AMDP), due to the specific cleavage by enterokinase.

FIG 2: Expressed and purified W19F variant. (A) 16.5 % Tricine SDS-PAGE. Lanes (1A): 3 µl of

Molecular weight protein markers (BioRad) (2A): 2µl of CSF before purification process, (3A): 2µl of

purified fusion protein DvnRV41-fP-W19F; (4A): digestion of 5 µl of DvnRV41-fP-W19F with

enterokinase EKMax (Invitrogen); (5A) 5 µl of purified variant W19F. (B) Western blotting against

DvnV41 polyclonal antibodies (45). Lanes (1B) 3 µl of fusion protein DvnRV41-fP-W19F after

purification and desalting; (2B) 3 µl of digested fP-DvnRV41-W19F, (3C) 3 µl of purified variant

W19.

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DvnRV41-fP TRX-(His)6-(Asp)4-Lys-DvnRV41

1 10 20 30 40

DvnV41 TKYYGNGVYCNSKKCWVDWGQASGCIGQTVVGGWLGGAIPGKC

DvnRV41 AMDPTKYYGNGVYCNSKKCWVDWGQASGCIGQTVVGGWLGGAIPGKC

W19F AMDPTKYYGNGVYCNSKKCWVD FGQASGCIGQTVVGGWLGGAIPGKC

Q21S AMDPTKYYGNGVYCNSKKCWVDWGSASGCIGQTVVGGWLGGAIPGKC

A22G AMDPTKYYGNGVYCNSKKCWVDWGQGSGCIGQTVVGGWLGGAIPGKC

S23Y AMDPTKYYGNGVYCNSKKCWVDWGQAYGCIGQTVVGGWLGGAIPGKC

V30F AMDPTKYYGNGVYCNSKKCWVDWGQASGCIGQTFVGGWLGGAIPGKC L35F AMDPTKYYGNGVYCNSKKCWVDWGQASGCIGQTVVGGWFGGAIPGKC A38V AMDPTKYYGNGVYCNSKKCWVDWGQASGCIGQTVVGGWLGGVIPGKC P40V AMDPTKYYGNGVYCNSKKCWVDWGQASGCIGQTVVGGWLGGAIVGKC

FIG 1

Rihakova et al. 2008

A

B

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FIG 2

Rihakova et al. 2008.

1 2 3 4 5 1 2 3

3640

2660

16 000

840

350

A B

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1 2 3 accepted - applied and environmental...

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