See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/234067738

Purification and Biochemical Characterization of a Highly Thermostable

Bacteriocin Isolated from Brevibacillus brevis Strain GM100

Article in Bioscience Biotechnology and Biochemistry · January 2013

DOI: 10.1271/bbb.120681 · Source: PubMed

CITATIONS READS

18 261

5 authors, including:

Ghadbane Mouloud Daoud Harzallah

University of Mohamed Boudiaf M’sila Ferhat Abbas University of Setif1
55 PUBLICATIONS 230 CITATIONS 95 PUBLICATIONS 867 CITATIONS

SEE PROFILE SEE PROFILE

Bassem Jaouadi
Centre of Biotechnology of Sfax (CBS)
131 PUBLICATIONS 3,196 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

biodiversity of prokaryotic microflora in salines environments of Algerian Sahara. Biomolecules products. View project

The needle-cast disease of Atlas cedar : Taxonomy and phylogenetics of its causal agent Lophodermium cedrinum View project

All content following this page was uploaded by Ghadbane Mouloud on 11 February 2016.

The user has requested enhancement of the downloaded file.

Biosci. Biotechnol. Biochem., 77 (1), 151–160, 2013

Purification and Biochemical Characterization of a Highly Thermostable

Bacteriocin Isolated from Brevibacillus brevis Strain GM100
Mouloud G HADBANE,1;2; y Daoud H ARZALLAH,1 Atef Ibn L ARIBI,3
Bassem JAOUADI,4 and Hani B ELHADJ1
1
Laboratory of Applied Microbiology, Department of Microbiology, Faculty of Natural and Life Sciences,
University Ferhat Abbas, Setif 19000, Algeria
2
Laboratory of Plant Biotechnology, Department of Natural and Life Sciences, Faculty of Sciences,
University of M’sila, PO Box 166, Chebilia, M’sila 28000, Algeria
3
Postharvest Technology Center, Valencian Institute for Agricultural Research, 46113 Moncada, Valence, Spain
4
Laboratory of Microorganisms and Biomolecules, Centre of Biotechnology of Sfax, University of Sfax,
Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax 3018, Tunisia

Received September 5, 2012; Accepted October 11, 2012; Online Publication, January 7, 2013
[doi:10.1271/bbb.120681]

A bacteriocin-producing (11,000 AU mL 1 ) strain was as bacteriocins. Traditional medicinal antibiotics are

isolated from the rhizosphere of healthy Algerian plants also produced by some bacteria, but these and bacter-
Ononis angustissima Lam., and identified as Brevibacil- iocins should be considered different antimicrobial
lus brevis strain GM100. The bacteriocin, called Bac- compounds. An important criterion of being a bacter-
GM100, was purified to homogeneity from the culture iocin is that bacteriocins are ribosomaly synthesized,
supernatant, and, based on MALDI-TOF/MS analysis, while antibiotics are made by multi-enzyme complexes.
was a monomer protein with a molecular mass of Most bacteriocins kill a narrow spectrum of bacteria as
4375.66 Da. The 21 N-terminal residues of Bac-GM100 compared to antibiotics. Moreover, most bacteriocins
displayed 65% homology with thurincin H from Bacil- are more potent against their target bacteria, while
lus thuringiensis. Bac-GM100 was extremely heat-stable higher concentrations of antibiotics are needed to kill the
(20 min at 120  C), and was stable within a pH range of target bacteria.4) Bacteriocins are produced by both
3–10. It proved sensitive to various proteases, which gram-positive and gram-negative bacteria, and the
demonstrated its protein nature. It was also found to bacteriocins of gram-positive bacteria appear to show
display a bactericidal mode of action against gram- a broader range of susceptible organisms.5) In fact, the
negative (Salmonella enteric ATCC 43972, Pseudomonas use of bacteria as biocontrol agents has been extensively
aeruginosa ATCC 49189, and Agrobacterium tumefa- studied in the last few decades, and a wide array of
ciens C58) and gram-positive (Enterococcus faecalis bioactive metabolites, including bacteriocins, antibiot-
ENSAIA 631 and Staphylococcus aureus ATCC 6538) ics, antifungals, antivirals, insecticides, herbicides, and
bacteria, and a fungistatic mode of action against the anticancer agents, has been reported in the literature.6,7)
pathogenic fungus Candida tropicalis R2 CIP 203. The efficiency of these biological agents has been
demonstrated in several fields.8,9)
Key words: Brevibacillus brevis; bacteriocin; purifica- Although gram-positive Bacillus species are often
tion; thermostability; bactericidal and considered promising biocontrol agents as effective as
fungistatic mode of action gram-negative bacteria, they have received much less
attention and are studied for their compounds production
The rapid rise and spread of multi-resistant microbial with potential uses in many fields.10) Above all, those
pathogens has forced a consideration of alternative produced by gram-positive bacteria have attracted much
methods of combating infection. Biological control of more interest, particularly for their promising agricul-
infectious diseases, a new emerging biological tool, tural potential, as they can open new opportunities for
offers a powerful alternative to conventional synthetic the alleviation of various pathogenic conditions. In
chemicals in human and animal health management as particular, Bacillus strains are known to be thermo-
well as in agricultural and food biotechnology.1) How- tolerant and spore-forming species that show rapid
ever, microbes produce an extraordinary array of micro- growth in liquid culture and are widely distributed in
bial defense systems. These include broad-spectrum soils. Various Bacillus species produce bacteriocins.
classical antibiotics, metabolic by-products (organic These include bacteriocin-like inhibitory substances
acids), lytic agents (lysozymes), numerous proteina- (BLISs)11) produced by B. cereus ATCC 4579, coagu-
ceous exotoxins, and bacteriocins.2,3) In the microbial lin12) produced by B. coagulans, and bacthuricin F4,13)
world, antimicrobial peptides are an important part of entomocin 110,14) thuricin 17 (T17)15) produced by
the defense system of bacteria, and they are referred to B. thuringiensis. However, and despite their promising
y
To whom correspondence should be addressed. Fax: +213-36-90-38-79; E-mail: mouloud ghadbane@yahoo.fr
Abbreviations: AU, activity unit; BSA, bovine serum albumin; CFUs, colony forming units; FPLC, fast protein liquid chromatography; IPTG,
isopropyl-thio-
-D-galactopyranoside; LB medium, Luria-Bertani medium; MALDI-TOF/MS, matrix assisted laser desorption ionization-time of
flight/mass spectrometry; O.D, optical density; SDS–PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis
152 M. GHADBANE et al.

properties as potential antibacterial agents, only a few peptone, 15; yeast extract, 5; and glucose, 10; supplemented with
Bacillus bacteriocins have been characterized to date. In 15 mL of adenine solution (2 g L1 ) at 30
C for 24 h in an orbital
incubator with shaking at 200 rpm. Strain GM100 was maintained as
addition, B. subtilis is known to produce many anti-
frozen stock at 80
C in LB broth containing 20% (v/v) glycerol.
microbial peptide substances, such as bacilysin16) and Before experimental use, the cultures were propagated twice in LB
subtilosin A.17) Iturine A, a mixture of homologous medium at 30
C for 12 h. The transfer inoculum was 1% (v/v).
cyclic peptides (A1 through A8 ), was also obtained from
B. subtilis.18) It has been found that iturine, secreting Identification of the Bacillus strain. The strain was examined for
B. subtilis NB22, can be used as an agent to control soil- gram reaction, cell shape, oxidase and catalase reactions, motility,
borne tomato diseases.19) Furthermore, Brevibacillus endospore formation, and oxygen relationships. An API 50 CH strip
brevis strains are considered probiotic agents both for (bioMérieux, Marcy-l’Etoile, France) was used to investigate the
physiological and biochemical characteristics of strain GM100 in
human and for animal use.20) Several molecular methods
accordance with the instructions of the manufacturer. The 16S rRNA
are available for the exact identification of a given gene was amplified by polymerase chain reaction (PCR) using two
bacterial strain and, 16S rDNA gene sequencing is the universal primers: forward, 50 -AGAGTTTGATCCTGGCTCAG-30 ,
first tool for this purpose.21) and reverse, 50 -AAGGAGGTGATCCAAGCC-30 , designed on the
During a search program for bioactive antibacterial basis of positions 8 to 27 and 1,541 to 1,525 respectively. These are
and antifungal compounds from Bacillus with anti- conserved zones within the rRNA operon of E. coli.21) The Genomic
Agrobacterium spp. activity, a new bacterium, GM100, DNA of strain GM100 was used as template for PCR amplification (35
cycles, 94
C for 30 s denaturation, 60
C for 1 min primer annealing,
was selected, and characterized by means of phenotypic and 72
C for 1.5 min extension). The amplified approximately 1.5-kb
and 16S rDNA gene sequencing. This paper describes PCR product was cloned in pGEM-T Easy vector (Promega, Madison,
the selection and identification of this bacterium. The WI), to obtain pGM100-16S plasmid (this study). E. coli DH5
purification and the biochemical characterization of a (F supE44 80 lacZ M15 (lacZYA-argF) U169 endA1 recA1
bacteriocin-like substance, Bac-GM100, from the super- hsdR17 (rk  , mk þ )deoR thi-1   gyrA96 relA1) (Invitrogen Life
natant culture of this new strain are reported. The paper Technologies) was used as host strain. All recombinant clones of
E. coli were grown in LB broth medium with the addition of
also further explores the inhibitory spectrum and mode
ampicillin, isopropyl-thio-
-D-galactopyranoside (IPTG), and X-gal
of action of Bac-GM100, and assesses its activity for screening. DNA electrophoresis, DNA purification, restriction,
against gram-positive and gram-negative bacteria as ligation, and transformation were all performed by a method
well as fungi. previously described by Sambrook et al.22)

Materials and Methods DNA sequencing and phylogenetic analysis. The nucleotide
sequence of the 16S rRNA gene was determined on both strands
Enzymes and chemicals. Bovine serum albumin (BSA), trypsin, using a BigDyeÒ Terminator v3.1 Cycle Sequencing Kit and an
pepsin, papain, pronase E, and proteinase K were purchased from automated DNA sequencer ABI PrismÒ 3100-Avant Genetic Analyser
Sigma Chemical (St. Louis, MO). Sephadex G-75 and Mono Q FPLC (Applied Biosystems). 16S rDNA sequence analysis was performed by
were from Pharmacia (Pharmacia, Uppsala, Sweden). A protein assay means of the BLAST program (www.ncbi.nlm.nih.gov/blast). Phylo-
kit was from Bio-Rad Laboratories (Hercules, CA). All other genetic and molecular evolutionary analyses were conducted by means
chemicals and reagents used were of analytical grade or the best of molecular evolutionary genetics analysis (MEGA) software version
grade commercially available, unless otherwise stated. 4.1. Distances and clustering were calculated by the neighbor-joining
method. Bootstrap analysis was used to evaluate the tree topology of
Bacterial strains and growth conditions. Strain GM100, exhibiting the neighbor-joining data by performing 100 re-samplings.23)
pronounced antagonistic activities, was isolated from the rhizosphere
of healthy plants Ononis angustissima Lam. grown in the region of Biological assay of antimicrobial activities. The ability of Brevi-
Biskra (in southern of Algeria) at a nursery. Five gram-positive bacillus brevis strain GM100 to produce diffusible metabolites was
bacteria (Micrococcus luteus LB 14110, Staphylococcus aureus ATCC assessed by Agar Well Diffusion Assay.24) The diameters of the
6538, Listeria ivanovii BUG 496, Enterococcus faecalis ENSAIA 631, inhibition zones were measured. The bacteriocin samples to be spotted
and E. faecalis JH 2-2), and 17 gram-negative bacteria (Escherichia were serially diluted two-fold, and the reciprocal of the highest dilution
coli ATCC 8739, Salmonella entericia ATCC 43972, S. typhimurium, showing inhibition was considered as one arbitrary activity unit (AU).
Pseudomonas savastanoi pv. savastanoi, P. aeruginosa ATCC 49189, Uninoculated culture broth was used as negative control.
A. tumefaciens S56, A. tumefaciens CIP 497-74, A. tumefaciens CIP
111-78, A. tumefaciens ATCC 23308T , A. tumefaciens CFBP 6625, A. Bac-GM100 purification procedure. Five hundred mL of modified
tumefaciens NCPPB 925, A. tumefaciens RV3, A. tumefaciens C58, A. LB culture medium, obtained after 48 h of cultivation, was centrifuged
tumefaciens O363, A. rhizogenes CFBP 2408T , A. larrymoorei AF3.44, for 30 min at 10;000  g to remove microbial cells. The supernatant
and A. vitis CFBP 2678T ) were used as indicator microorganisms in containing extracellular active compounds was retained as crude
the antibacterial activity assays. Antifungal activity was determined bacteriocin preparation for subsequent assays. The crude extract was
against the Fusarium sp. and Candida tropicalis R2 CIP 203. precipitated with ammonium sulphate at a concentration from 40–60%.
Bac-GM100 from Brevibacillus brevis strain GM100 was produced The pellet obtained after centrifugation for 30 min at 10;000  g was
at 30
C for 48 h in an orbital incubator with shaking at 250 rpm, using dissolved in a minimum of 25 mm Tris–HCl at pH 8 (buffer A) and
a modified Luria-Bertani (LB) medium (in g L1 ): peptone, 15; yeast dialyzed overnight against repeated changes of the same buffer using
extract, 3; and NaCl, 2.5 (pH 7.4), supplemented with (in g L1 ): benzoylated membranes with a molecular weight cut-off of 1200 Da
.
glucose, 7; K2 HPO4 , 1; KH2 PO4 , 1; and MgSO4 7H2 O, 1). To (Sigma Chemical, St. Louis, MO). The clear supernatant was incubated
measure antibacterial activities, indicator microorganisms were grown for 2 h at 90
C. After rapid cooling, insoluble denatured proteins were
overnight in LB medium (pH 7.2) under aerobic conditions at 30
C for removed by centrifugation for 30 min at 10;000  g. The protein
the A. tumefaciens strains, A. rhizogenes, P. savastanoi pv. Savasta- solution, designated fraction I, was loaded on a 2:5  50 cm Sephadex
noi, P. aerigunosa ATCC 49189, E. coli ATCC 39, and Xanthomonas G-75 column equilibrated with buffer A. Elution of the protein was
compestris, and aerobically at 37
C for E. coli ATCC 8739 and done with the same buffer at rate of 45 mL h1 . The protein
S. aureus ATCC 6538. To determine antifungal activities, Fusarium concentration was monitored at 280 nm. Fractions were tested for
sp. was grown in Potato Dextrose Agar (PDA) for 7 d at 30
C. Spores bacteriocin activity. The active fraction, designated fraction II, was
were collected in sterile distilled water and then concentrated to collected and injected into a Mono Q column (2:6  20 cm) attached to
produce a suspension of approximately 104 spores mL1 . C. tropicalis an FPLC system previously equilibrated with buffer A. After washing
R2 CIP 203 was grown in YP10 medium containing (in g L1 ): of the column, no bacteriocin activity was detected in the washing
 Characterization of a Highly Thermostable Bacteriocin 153
solution. The adsorbed material was eluted with a linear NaCl gradient various organic solvents with different Log p values (chloroform,
(500 mL of 0–500 mm in buffer A) at a rate of 40 mL h1 . Fractions butanol, isopropanol, ethyl acetate, and methanol) at a final concen-
were collected automatically by the system and assayed for antago- tration of 60% (v/v). After incubation for 72 h at room temperature, the
nistic activity using A. tumefaciens C58 as indicator bacterium. organic solvent was evaporated in a vacuum concentrator and residual
Fractions containing active Bac-GM100 that eluted between 180 and antimicrobial activity was determined. The antimicrobial activity of
220 mm NaCl (fraction III) were collected and stored at 20
C in Bac-GM100, which was resuspended in buffer A without added
glycerol 20% (v/v) solution for further analysis. solvents, was used as control. To determine the sensitivity of a number
of enzymes on active Bac-GM100, a variety of proteolytic enzymes
Molecular mass determination, mass spectrometry, and amino acid were added at a final concentration of 1 mg mL1 . All the enzymes
sequencing. The active fraction eluted after each purification step was were dissolved in buffers, as recommended by the provider company.
pooled and subjected to sodium dodecyl sulphate-polyacrylamide gel The proteases used were trypsin, pepsin, papain, pronase E, and
electrophoresis (SDS–PAGE) using 5% stacking gel and 15% resolv- proteinase K. Following 48 h of incubation at 37
C, enzyme activity
ing gel, as reported by Laemmli.25) The protein concentration was was stopped by heating at 100
C for 5 min, and the remaining
measured as described by Bradford.26) Protein bands were visualized bacteriocin activity was measured in AU mL1 . Untreated bacteriocin
by Coomassie Brilliant Blue R-250 staining. Unstained #BM201 Low plus buffers, buffers alone, and the enzyme solutions were used as
Range Protein Marker (Bio Basic, Markham, Canada) was used as controls.
standard (in kDa): BSA, 66; ovalbumin, 45; porcine pepsi, 35;
triosephosphate isomerase, 27; trypsin inhibitor, 20; lysozyme, 14.4; Inhibitory spectrum and mode of action of Bac-GM100. The
parathyroid (1–84), 9.5; aprotinin, 6.5; and parathyroid (1–34), 4.5. inhibitory spectrum was checked by Agar Well Diffusion Assay.24)
Zymogram analysis was performed as previously described.27) The Pure bacteriocin, Bac-GM100 (500 AU mL1 ), was added to 200 mL
molecular mass of the purified Bac-GM100 was determined by of LB culture of A. tumefaciens C58 and 200 mL of YP10 of
MALDI-TOF/MS using a Voyager DE-RP instrument (Applied C. tropicalis R2 CIP 203 at the early exponential phase
Biosystems, Framingham, MA). To determine the N-terminal sequence, (107 CFU mL1 ). The two indicator microorganisms growing in LB
the protein band from the SDS gel was transferred to a Problott (A. tumefaciens C58) and YP10 (C. tropicalis R2 CIP 203) medium in
membrane (Applied Biosystems, Foster City, CA). Automated Edman the absence of bacteriocin were used as controls. Changes in the
protein degradation was performed with a protein sequencer (Protein turbidity of the cultures were recorded at an optical density (O.D) of
sequencer ABI Procise 492/610A, Applied Biosystems). 600 nm, and the number of viable cells (Colony forming units
CFU mL1 ) was determined by plaiting the samples on LB or YP10
Effects of pH and temperature on the Bac-GM100 bacteriocin. The agar at various time intervals.
pure bacteriocin Bac-GM100 (500 AU mL1 ) was resuspended in
various buffer solutions at 50 mm, ranging from pH 2 to 11 (citrate for Phytotoxicity effect of Bac-GM100 on tomato and muskmelon.
pH 2–6; phosphate for pH 7; Tris–HCl for pH 8–9, and glycine–NaOH Phytotoxicity was assessed by determination of the germination index
at 2 times, 72–96 h for tomato (Lycopersicon esculentum) and 30–60 h
for pH 9–11), and incubated for 48 h at 4
C. The antimicrobial activity
of Bac-GM100 was expressed in terms of AU mL1 , and was for muskmelon (Cucumis melo) by the standard method of Zucconi et
compared with the untreated controls. The thermal stability of Bac- al.28) Tomato and muskmelon seeds were treated with water (negative
GM100 was monitored at various temperatures. Accordingly, Bac- control), sodium hypochlorite 10% (w/v) (positive control), and two
different doses of Bac-GM100 (50 and 500 AU mL1 ). The post-
GM100 was resuspended in 25 mm Tris–HCl buffer at pH 8, subjected
to boiling temperatures (70, 80, 90, and 100
C for 2 h), and autoclaved germinated seeds were transplanted in a sterile potting mix as
(120
C, 20 min) prior to activity assay. described below. Traditional seed vigor biomarkers were determined
based on the total number of seedlings that emerged fully, shoot height,
shoot weight, and root length.
Antimicrobial activity and sensitivity to proteases, detergents, and
organic solvents. In order to determine the biological nature of the
Statistical analysis. The trial was established according to a
antimicrobial activity produced by the strain GM100, 1 mL of cell-free
randomized plots experimental design with three triplicates, including
supernatant was incubated for 3 h at 37
C in the presence of
30 plants in each replicate. The data were subjected to analysis of
1 mg mL1 of catalase (Boehringer, Germany). The untreated bacter-
variance using the Statistical Package for the Social Sciences (SPSS
iocin-containing cell-free supernatant served as control. Sensitivity to
V.11; SPSS, Chicago, IL). Mean values among treatments were
proteolytic enzymes of antimicrobial compounds was investigated by
compared by Duncan’s multiple range test at the 5% (p ¼ 0:05) level
the addition of trypsin, pepsin, papain, pronase E, and proteinase K at a
of significance.
final concentration of 1 mg mL1 to the culture supernatants of strain
GM100 (30
C, 48 h). The samples were incubated for 2 h at 37
C, and
immediately afterwards, residual activity was determined as described Nucleotide accession number. The 16S rRNA nucleotide sequence
above. The detergents used were Tween 40, Tween 60, Triton X-100, data for newly isolated Brevibacillus brevis strain GM100 was
submitted to the NCBI GenBank database, and was assigned accession
SDS, and urea at final concentrations of 15% (w/v), and ethylene-
diaminetetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride no. JX524820.
(PMSF), which were added to the culture supernatants of strain GM100
(30
C, 48 h) at a final concentration of 15% (v/v) and the reaction was Results and Discussion
incubated at 30
C for 2 h. The culture supernatants of the strain
GM100 (30
C, 48 h) were mixed with various organic solvents of Identification and molecular phylogeny of the micro-
different Log p values (chloroform, butanol, isopropanol, ethyl acetate, organism
and methanol) at a final concentration of 60% (v/v). After incubation
for 1 h at room temperature, the organic solvent was evaporated
Identification of the newly isolated bacterium
in a vacuum concentrator, and residual antimicrobial activity was (GM100) was based on both phenotypic and molecular
determined. methods. Morphological, biochemical, and physiologi-
cal characteristics, by the methods described in Bergey’s
Stability of the purified Bac-GM100 bacteriocin. The effect of Manual of Systematic Bacteriology,29) indicated that
detergent on bacteriocin activity and stability was investigated. This the isolated strain appeared in a bacillus form, aerobic,
involved the incubation of pure Bac-GM100 in the presence of a broad endospore-forming, gram-positive, catalase-positive,
range of detergents, (Tween 40, Tween 60, Triton X-100, SDS, and oxidase-positive, and motile. The carbohydrate profile
urea) at a final concentration of 15%. In this case, bacteriocin Bac-
GM100, which was resuspended in buffer A without any denaturing
of the isolate was also investigated using API 50 CH
agents, was used as control. Each sample and each control was strips. The results indicated that while the microorgan-
incubated at 37
C for 48 h. Activity was assayed using A. tumefaciens ism could utilize D-glucose, D-fructose, maltose, glyc-
C58. The purified Bac-GM100 (50 AU mL1 ) was also mixed with erol, D-mannitol, and D-ribose, could not utilize
154 M. GHADBANE et al.

89 Bacillus clausii strain MG17 (JQ313203)

87 Bacillus clausii strain 221-B (AB201793)
68 Bacillus clausii strain NSB10(FJ189774)

99 Bacillus clausii strain DSM 8716 (NR026140)

61 Bacillus clausii DSM 8716 (X76440)

97 Bacillus clausii strain O/C (AJ297491)

99 Bacillus subtilis strain PCL1612 (DQ779885)
Bacillus subtilis (DQ187378)
99 Bacillus sp. CO16 (DQ643042)

99 Bacillus pumilus (DQ988522)

83 Bacillus pumilus strain CBS (EF113957)

78 Bacillus sp. strain Biosubtyl (AF142575)
Brevibacillus brevis strain DSM 6472 (AB112717)
80 Brevibacillus brevis strain GM100 (JX524820)
99 Brevibacillus brevis strain NBRC 100599 (AB681205)

95
Brevibacillus brevis strain JCM 2503 T (D78457)
Brevibacillus brevis strain DSM 30 T (AB101593)
81
Brevibacillus brevis strain DSM 5619 (AB112730)
70 Brevibacillus brevis strain DSM 5760 (AB112731)

Escherichia coli ATCC 11775 T (X80725)

0.02

Fig. 1. Phylogenetic Tree Based on 16S rRNA Gene Sequences Showing the Position of Strain GM100 within the Radiation of the Genus Bacillus.
The sequence of E. coli ATCC 11775T (X80725) was chosen arbitrarily as outgroup. Bar, 0.02 nt substitutions per base. Numbers at nodes
(>50%) indicate support for the internal branches within the tree obtained by bootstrap analysis (percentages of 100 bootstraps). NCBI accession
numbers are presented in parentheses.

D-mannose, D-tagatose, L-arabinose, myo-inositol, raffi- to detergents caused about 25% inactivation of the
nose, erythritol, or adonitol. All the data obtained with antimicrobial activity for the SDS and urea agents, and
regard to the physiological and biochemical properties about 10% for the other surfactants tested (Tween 40,
of the isolate, therefore, strongly confirmed that strain Tween 60, Triton X-100, EDTA, and PMSF). As for
GM100 belonged to the Bacillus genus. treatment with the organic solvents (ethyl acetate,
The 16S rRNA gene sequence obtained was submitted methanol, and isopropanol), no significant alteration of
to GenBank BLAST search analysis, which yielded a antimicrobial activity produced by the strain GM100
strong homology, of up to 98%, with those of several was observed.
cultivated strains of Brevibacillus. The most similar
Brevibacillus strains identified by the BLAST analysis Bac-GM100 purification and molecular weight deter-
were Brevibacillus brevis strain NBRC 100599 (acces- mination
sion no. AB681205) and Brevibacillus brevis strain Contaminating proteins in the cell-free supernatant
DSM 6472 (accession no. AB112717). These sequences were removed, and then the Bac-GM100 in the super-
were imported into the MEGA software and aligned. A natant was concentrated by ammonium sulphate precip-
phylogenetic tree was constructed (Fig. 1), and the itation (40–60%, w/v) and heat treated for 2 h at 90
C
findings further confirmed that strain GM100 (accession (fraction I), followed by Sephadex G-75 column
no. JX524820) was closely related to those of the chromatography (fraction II). In order to obtain a highly
Brevibacillus strains. In summary, all the results purified protein, fraction II was subjected to ion
obtained strongly suggested that this isolate ought to exchange chromatography. At this step, one major
be identified as Brevibacillus brevis strain GM100. absorbance symmetrical peak, fraction III, was eluted
at a 200-mm salt gradient. The purification process is
Effects of proteases, detergents, and organic solvents summarized in Table 1. Bacteriocin purity was estimated
on antimicrobial activity to be about 41-fold greater than that of the crude extract.
Enzymatic tests showed that the antimicrobial activity The presence of the inhibition zone determined after
against three tested indicator microorganisms, A. tume- 24 h of incubation under appropriate conditions revealed
faciens C58, L. ivanovii BUG 496, and C. tropicalis R2 that the purified bacteriocin Bac-GM100 exhibited a
CIP 203, of the supernatant culture of the strain GM100 broad inhibitory spectrum against all the pathogenic
was not affected by the addition of catalase, indicating indicator strains of the gram-positive and gram-negative
that the growth inhibition observed was not due to bacteria tested (Table 2). The yield of the purified
hydrogen peroxide production. In contrast, treatment bacteriocin preparation was about 23% based on total
with the proteolytic enzymes (trypsin, pepsin, papain, activity, and the specific activity was 84,013 AU mg1 .
pronase E, and proteinase K) caused complete inactiva- Electrophoresis under denaturing conditions (SDS-
tion of the antimicrobial compounds, which thus PAGE) revealed a single monomeric protein band at a
identified them as proteinaceous substances. Exposure molecular mass estimated to be 4.5 kDa (Fig. 2A). At
 Characterization of a Highly Thermostable Bacteriocin 155
Table 1. Flow Sheet for the Purification of Bacteriocin Bac-GM100 from Brevibacillus brevis Strain GM100

Total activity Total protein Specific activity Yield Purification

Purification step
(AU)a; 103 (mg)b; (AU mg1 ) (%) (factor)
Crude extract 5;500:00  4:75 2;678:17  422 2,053 100 1
(NH4 )2 SO4 Fractionation (40–60%) 4;125:18  3:49 223:26  76 18,477 75 9
Heat treatment (2 h at 90
C) 3;846:52  2:44 62:48  10 58,743 61 28
Sephadex G-75 2;165:10  1:34 32:95  7 65,708 39 32
Mono Q Sepharose FPLC 1;269:44  1:11 15:11  3 84,013 23 41
a
Antibacterial activity in arbitrary units (AU) was assayed by Agar Well Diffusion Assay using A. tumefaciens C58 as indicator strain.
b
Amounts of protein were estimated by the Bradford method.26)

Values represent means of three replicates, and  standard errors are reported.

Table 2. Antagonistic Spectrum of Bac-GM100

Indicator strains Diameter of inhibition zone (mm)b

Gram-positive bacteria Micrococcus luteus LB 14110 18  1:6
Staphylococcus aureus ATCC 6538 30  3:0
Listeria ivanovii BUG 496 31  3:5
Enterococcus faecalis ENSAIA 631 33  3:3
Enterococcus faecalis JH 2-2 25  2:0
Gram-negative bacteria Escherichia coli ATCC 8739 14  1:1
Salmonella enterica ATCC 43972 36  3:8
Salmonella typhimurium 20  2:0
Pseudomonas savastanoi pv. savastanoi 19  1:8
Pseudomonas aeruginosa ATCC 49189 34  3:5
Agrobacterium tumefaciens/Biovar 1 complexa S56 (genomovar G1) 24  2:0
CIP 497-74 (genomovar G2) 23  2:0
CIP 111-78 (genomovar G3) 25  2:0
ATCC 23308T (genomovar G4) 30  3:5
CFBP 6625 (genomovar G5) 23  2:0
NCPPB 925 (genomovar G6) 20  2:0
RV3 (genomovar G7) 22  2:0
C58 (genomovar G8) 35  3:5
O363 (genomovar G9) 25  2:0
Agrobacterium rhizogenes CFBP 2408T 15  1:5
Agrobacterium larrymoorei AF3.44 18  1:6
Agrobacterium vitis CFBP 2678T 25  2:0
Fungi Candida tropicalis R2 CIP 203 35  3:6
Fusarium sp. 21  2:0
The inhibitory level was estimated by measuring the diameter of the inhibition zone of the indicator strain.
ATCC, American Type Culture Collection (Manassas, VA)
CIP, Collection of the Pasteur Institute (France)
CFBP, French Collection of Bacterial Phytopathogenes, INRA (Angers, France)
a
The taxon Agrobacterium tumefaciens assimilated to the biovar 1 of Agrobacterium spp. is a complex of at least nine different genomic species or genomovars.47)
b
Values represent means of three replicates, and  standard errors are reported.

4.5 kDa, the antibacterial activity of this single band //www.expasy.ch/sprot/) using BLASTP search soft-
protein was confirmed by overlaying it on indicator ware. It was found that this bacteriocin contained a
strain A. tumefaciens C58, which revealed a growth unique sequence, suggests that it is of a novel com-
inhibitory zone at the position viewed in the stained gel pound. It showed 65% homology with bacteriocin
(Fig. 2B). Mass spectrometry analysis indicated that thurincin H from B. thuringiensis SF361 (accession
Bac-GM100 had a molecular mass of 4,375.66 Da no. 2LBZ A).
(Fig. 2C). The purified Bac-GM100 bacteriocin exhib-
ited single symmetrical elution peaks corresponding to a Biochemical properties of purified bacteriocin
protein of nearly 4.5 kDa according to gel filtration In order to determine the thermostability of Bac-
chromatography (Fig. 2D). GM100, inhibitory activity was examined following
exposure to a variety of temperatures. No activity loss
N-Terminal sequence of purified Bac-GM100 was detected following 2 h of exposure to a temperature
The first 21 N-terminal amino acid residues of the range of up to 100
C or 20 min of treatment at 120
C for
blotted purified antimicrobial peptide Bac-GM100 from autoclaving. Moreover, low temperature storage (20
Brevibacillus brevis strain GM100 were determined to and 4
C for 72 h), did not appear to alter the activity of
be DWTFANWSCLVCDDCSVNLTY. This sequence the purified protein. Bac-GM100 retained its biological
was subjected to comparison with protein sequences in activity within pH 3–10, but activity was drastically
the GenBank non-redundant nucleotide database (http: reduced at pH 12. In contrast, bacthuricin F4 from
//www.ncbi.nlm.nih.gov/) using the BLASTP and B. thuringiensis preserved only 20% of its activity after
tBlastn search programs. It was further subjected to incubation at 90
C for 30 min.13) There are reports in the
comparison through the Swiss-Prot database (http: literature that many bacteriocins lose their heat stability
156 M. GHADBANE et al.

A B C
MM
(kDa) M 1 2
4375.66 Da
66
45
35
27

Relative intensity (%)

20

14.4
9.5
6.5
4.5
16414.0 24914.4 3414.0 4444.2 5423.4 6401.0
Mass (M/Z)

2,5 12,000
D

Bac-GM100 activity (AU mL )

-1
10,000
2
Absorbance at 280 nm

8,000
1,5

6,000

1
4,000

0,5
2,000

0 0
1 5 9 13 17 21 25 29 33 37 41 45

Fraction number

Fig. 2. Chromatography on Sephadex G-75, Electrophoretic, Zymograms, and Mass Spectrometry Analyses of the Purified Bac-GM100.
Lane M, Low Range Protein Marker (Bio Basic, Markham, Canada). Lane 1, purified Bac-GM100 (30 mg of protein) (A). Lane 2, portion of
the renaturated SDS–PAGE, overlaid on A. tumefaciens C58 as indicator strain (B). MALDI-TOF spectrum of 30 pmol purified Bac-GM100.
The mass spectrum showed a series of multiply protonated molecular ions. The molecular mass of Bac-GM100 was found to be 4,375.66 Da (C).
Elution and activity of the bacteriocin (D).

upon purification.13) In contrast, purified Bac-GM100 logarithmic growth phase resulted in a rapid decrease
retained its full heat stability, as has been reported for in the number of A. tumefaciens C58 viable cells (from
bacteriocin Bac 14B from B. subtilis strain 14B.30) 107 CFU mL1 to less than 102 CFU mL1 ) over a
Exposure to various detergents showed that only period of 5 h (Fig. 3A). The optical density readings
Tween 40 and Tween 60 eliminated the bacteriocin for this indicator microorganism remained constant after
activity of Bac-GM100. Treatment with Triton X-100, the addition of Bac-GM100. Our results indicate that the
SDS, and urea were, however, found to have no effect on bacteriocin studied has a bactericidal effect against the
the bacteriocin activity of Bac-GM100. Moreover, A. tumefaciens C58 strain. As for C. tropicalis R2 CIP
treatment with organic solvents (isopropanol, chloro- 203, the optical density readings were very similar in
form, and ethyl acetate) was found to have no significant presence and the absence of Bac-GM100 (Fig. 3B), but,
alteration effect on the activity of Bac-GM100. Meth- we noticed a decrease in the number of viable cells
anol and butanol, on the other hand, slightly affected the grown in the presence of bacteriocin GM100 at 42 h of
bacteri- growth. These data indicate that Bac-GM100 acts with
ocin activity of Bac-GM100. When treated with various a fungistatic effect against C. tropicalis R2 CIP 203
hydrolytic enzymes, purified bacteriocin Bac-GM100 (Fig. 3B).
was to undergo significant decreases in inhibitory action Bac-GM100 can be considered the second antimicro-
after treatment with proteases, showing that the bacter- bial protein to show a spectrum of action that is
iocin has a proteinaceous nature. This bacteriocin activity particularly inhibitory against Agrobacterium spp.
was similar to that of bacthuricin F4 from B. thurin- strains, which are responsible for the induction of
giensis13) and Bac 14B from B. subtilis strain 14B.27,30) neoplasic diseases, after bacteriocin Bac 14B from
B. subtilis strain 14B, which shows a broad inhibitory
Inhibitory spectrum and mode of action of purified spectrum of antagonistic activities against pathogenic
Bac-GM100 indicator strains of gram-positive and negative bacte-
The addition the purified Bac-GM100 at 500 ria.27) Purified bacteriocin Bac-GM100 exhibited a wide
AU mL1 to cells of A. tumefaciens C58 (4 h old) and spectrum of inhibitory action. In fact, it inhibited both
C. tropicalis R2 CIP 203 (8 h old) in the early gram-positive and negative bacteria. The general mode
 Characterization of a Highly Thermostable Bacteriocin 157
10 1,4
A Addition of
9
Bac-GM100
1,2
8

Absorbance at 600 nm
7 1

Log 10 (CFU mL )
-1
6
0,8
5
0,6
4

3 0,4

2
0,2
1

0 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14

Incubation time (h)

9,5 5
B
4,5
9
4

Absorbance at 600 nm
8,5 3,5
Log 10 (CFU mL )
-1

3
8
2,5
7,5
2

7 1,5

1
6,5
0,5

6 0
0 4 8 10 12 16 20 24 28 32 36 40 44

Incubation time (h)

Fig. 3. Effect of Bac-GM100 on the Growth of A. tumefaciens C58.

Optical density at 600 nm in absence ( ) and the presence ( ) of Bac-GM100. Viable cell counts (CFU mL1 ) in absence ( ) and the presence
( ) of Bac-GM100 (A). Effect of Bac-GM100 on the growth of C. tropicalis R2 CIP 203. Optical density at 600 nm in the absence ( ) and the
presence ( ) of Bac-GM100. Viable cell counts (CFU mL1 ) in the absence ( ) and presence ( ) of Bac-GM100 (B).

of action of bacteriocin has been reported to be particular, Nisin (3.35 kDa) is a natural, toxicologically
bactericidal due to the pore-forming action of the cell safe, antibacterial food preservative. It is regarded as
membrane.31,32) Several large (>10 kDa) streptococcal natural because it is a polypeptide produced by certain
antimicrobial proteins have a narrow spectrum of strains of the food-grade lactic acid bacterium Lacto-
action.33) The inhibitory spectrum of, for instance, coccus lactis subsp. lactis during fermentation. It
dysgalacticin (21 kDa) is fairly narrow, and is limited exhibits antimicrobial activity towards a wide range of
to strains of Lancefield serogroups A, C, and G. The gram-positive bacteria, and is particularly effective
range of organisms inhibited by SA-M57 (17 kDa), on against spores. It shows little or no activity against
the other hand, is unusual, consisting mainly of non- gram-negative bacteria, yeasts, or moulds.38) Cerein
streptococcal gram-positive species, including Micro- MRX1 (3.13 kDa), produced by B. cereus strains, shows
coccus luteus, Lactococcus lactis, and six species of a bactericidal mode of action against bacteria of concern
Listeria, B. megaterium, and S. simulans. Stellalysin is in the food industry, such as species of Bacillus
another 29-kDa bacteriocin, with an inhibitory spectrum (including B. cereus, B. coagulans, B. subtilis, B. pum-
that includes S. pyogenes, S. gordonii, and S. mutans.34) ilus, and others) and L. innocua39) Brevicin AF01,
The present investigation of Bac-GM100 also indicated produced by B. brevis AF01, isolated from a wheat
that it has a bactericidal effect on the indicator, field shows a bactericidal mode of action against several
A. tumefaciens C58, and functions as a fungistatic MRSA strains.40) Thuricin 17 (3.16 kDa) is produced by
against C. tropicalis R2 CIP 203. Some bacteriocins, the plant growth-promoting rhizobacterium strain
such as thoeniicin, that have very limited spectra among B. thuringiensis NEB17, isolated from soybean root
tested lactic bacteria in spite of a small MW (7.13 kDa), nodules in Quebec, Canada. It is active against B. thur-
are reported to be bactericidal on one strain and ingiensis, B. cereus strains, and related Bacilli, as well
bacteriostatic on others.35) However, a few bacteriocins as gram-negative strain E. coli MM294 (pBS42),15,41)
have been reported to be active against gram-negative whereas, thurincin H (3.14 kDa), produced by B. thur-
bacteria, and particularly against fungi.36) Most of the ingiensis, acts on Bacillus, Listeria, and Carnobacte-
bacteriocins described as antifungal agents from Lacto- rium, but does not kill gram-negative bacteria.42)
bacillus are organic compounds or diketopiperazine Bacteriocin ST15 (3.94 kDa) is a peptide produced by
derivatives of molecular masses lower than 1 kDa.37) In E. mundtii that exhibits a broad spectrum of antimicro-
158 M. GHADBANE et al.

150 Tomato (Lycopersicon esculentum)

A 72 h 96 h

Index of germination (%)

125

100

75
a
b
50 A
B b b
25 B B
0
Control Sodium hypochlorite Bac-GM100 Bac-GM100
(10% w/v) (50 AU mL-1) (500 AU mL-1)
Treatments

150 Muskumelon (Cucumis melo)

B 30 h 60 h
Index of germination (%)

125

100

75 d
b D
50 d
B c
25
C A
0
Control Sodium hypochlorite Bac-GM100 Bac-GM100
(10% w/v) (50 AU mL-1) (500 AU mL-1)
Treatments

Fig. 4. Germination Indices of Tomato (Lycopersicon esculentum) (A) and Muskmelon (Cucumis melo) (B) Determined in Distilled Water
(Control), Sodium Hypochlorite (10% w/v), and Bac-GM100 at Various Concentrations.
Duncan’s test (p ¼ 0:05) indicated that the histograms with different letters (a, A, b, B, c, C, d, D) are significantly different. Values are means
of three independent experiments, and error bars indicate the standard deviation.

bial activity against gram-positive and negative bacte- sented similar ratios of germination as compared to the
ria.43) Sakacin C2 (5.5 kDa) displayed broad antimicro- control subjected to water treatment (Fig. 4). Applica-
bial activity not only against a larger range of LAB but tion of Bac-GM100 (50 AU mL1 ) was observed to
also against many gram-positive and negative bacteria. improve the germinative energy of the seeds and to
The inhibitory spectra of known bacteriocins from entail no inhibitory effect on seed germination as
L. sakei are quite narrow. For example, the inhibition compared to the control subjected to water treatment.
spectrum of sakacin G appeared to be limited to L. sakei The indices of germination at the half-period were 36
and Pediococcus cerevisiae of the tested strains, as far as and 25 h for tomato (Fig. 4A) and muskmelon (Fig. 4B)
LABs are concerned. Among three kinds of bacteriocins respectively, significantly higher than for the negative
(sakacins P, 5X, and 5T) produced by L. sakei 5, only controls (Fig. 4). The germinative energy yields ob-
sakacin 5X inhibited many gram-positive beer-spoilage tained for treatment with Bac-GM100 (500 AU mL1 )
organisms, such as L. brevis, and E. facealis, but their and sodium hypochlorite, on the other hand, were found
inhibitory activity towards gram-negative bacteria has to be lower than the optimum (ratio < 50%). The initial
not been determined yet, while sakacin P and sakacin 5T vigor response was measured using traditional agro-
inhibited only of the two out of 13 LAB strains.44) These nomic parameters, germination percentage, root length,
behaviors clearly indicate that there is no relationship and shoot weight and height. The results indicated that
between the molecular size of bacteriocins and their the vigor response with respect to Bac-GM100
spectra. (50 AU mL1 ) was higher than those obtained for the
other treatments (Fig. 5). Nevertheless, Bac-GM100
Phytotoxicity effect of Bac-GM100 on plant species (500 AU mL1 ) has have a negative effect on the vigor
Seed germination of tomato and muskmelon was response of the seeds, bringing about a significant
performed with two different concentrations of Bac- reduction in root length (Fig. 5A) as well as shoot height
GM100 (50 and 500 AU mL1 ), and the germination (Fig. 5B) and weight (Fig. 5C). These results are
ratios were compared to those obtained for seeds treated comparable to those obtained for sodium hypochlorite
with water (negative control) and those treated with 10% treatment, where the seed vigor biomarkers were
(w/v) sodium hypochlorite (positive control). The drastically affected (data not shown).
results indicated that seed germination was strongly The findings of the current study also indicate that the
inhibited for the two species studied when treated with tomato and muskmelon seedlings treated with Bac-
sodium hypochlorite and Bac-GM100 (500 AU mL1 ). GM100 (50 AU mL1 ) showed higher and average
Bac-GM100 (50 AU mL1 ) did not show any inhibitory germination rates as compared to the Bac-GM100
effect on seed germination, and the two crops repre- solution (500 AU mL1 ) and the control seedlings
 Characterization of a Highly Thermostable Bacteriocin 159

A 225 Muskmelon Tomato

200

Shoot weight (g)

175
A a
150
125
100
A
a D
75
d
50 B
25 c
0
Control Sodium hypochlorite Bac-GM100 Bac-GM100
(10% w/v) (50 AU mL-1) (500 AU mL-1)

Treatments

25
B Muskmelon Tomato

20
Shoot height (cm)

15
a
10
A

5
a
0
B C b D d
Control Sodium hypochlorite Bac-GM100 Bac-GM100
(10% w/v) (50 AU mL-1) (500 AU mL-1)

Treatments

25
C Muskmelon Tomato

20
Root length (cm)

15
a
10
A
5
A b B c D d
0
Control Sodium hypochlorite Bac-GM100 Bac-GM100
(10% w/v) (50 AU mL-1) (500 AU mL-1)

Treatments

Fig. 5. Effects of the Germination with Distilled Water (Control), Sodium Hypochloride (10% w/v), and Bac-GM100 at Concentrations of 50 and
500 AU mL1 on Shoot Weight (A), Shoot Height (B), and Root Length (C) at 2 Months after Transplantation of Tomato and Muskmelon Plants.
Duncan’s test (p ¼ 0:05) indicated that the histograms with different letters (a, A, b, B, c, C, d, D) are significantly different. Values are means
of three independent experiments, and error bars indicate the standard deviation.

respectively. Germinative energy can play an important Acknowledgments

role in the achievement of quick uniform seedling
emergence and the reduction of damping-off incidence, This work was funded by the Algerian Ministry of
thus improving yield.45) It has been reported that the use Higher Education and Scientific Research. We wish to
of fungicides is effective in enhancing germination, express our gratitude to Dr. Bouazza Lyas, Dr.
emergence, and growth and in reducing damping-off. In Boufennara Souhil (University of Mentouri, Constantine,
addition, accelerated germination has been reported to Algeria), and Dr. Abdelhamid Beji (Max-Planck-Institut,
help improve stress resistance and enhance overall plant Munich, Germany) for constructive discussion and
growth and productivity.46) This might account for the valuable help during this study. Special thanks are due
usefulness of the Bac-GM100 solution for seed dis- also to Professor Anouar Smaoui, of the English Depart-
infection. ment of the Sfax Faculty of Science for carefully
proofreading and polishing the language of the present
paper.
 160 M. GHADBANE et al.

References 25) Laemmli UK, Nature, 227, 680–685 (1970).

26) Bradford MM, Anal. Biochem., 72, 248–254 (1976).
1) Ojiambo PS and Scherm H, Phytopathology, 96, 1168–1174 27) Hammami I, Rhouma A, Jaouadi B, Rebai A, and Xavier N,
(2006). Lett. Appl. Microbiol., 48, 253–260 (2009).
2) Harzallah D, Sadallah S, and Larous L, Commun. Agric. Appl. 28) Zucconi F, Forte M, Monac A, and Beritodi M, Biocycle, 22,
Biol. Sci., 69, 443–447 (2004). 27–29 (1981).
3) Messaadia N and Harzallah D, World Acad. Sci. Eng. Technol., 29) Yabuuchi E, Kansenshogaku Zasshi, 75, 653–655 (2001).
73, 904–908 (2011). 30) Hammami I, Jaouadi B, Ben Bacha A, Rebai A, Bejar S, Nesme
4) Zhang Y, Zhang L, Du M, Yi H, Guo C, Tuo Y, Han X, Li J, X, and Rhouma A, Biotechnol. Bioproc. Eng., 17, 41–49 (2012).
and Yang L, Microbiol. Res., 167, 27–31 (2011). 31) Zhou W, Liu GR, Li PL, Dai YQ, and Zhou K, Wei Sheng Wu
5) Riley MA and Wertz JE, Ann. Rev. Microbiol., 56, 117–137 Xue Bao, 47, 260–264 (2007).
(2002). 32) Deraz SF, Karlsson EN, Khalil AA, and Mattiasson B, J. Ind.
6) Gillor O and Ghazaryan L, Recent Pat. Antiinfect. Drug Discov., Microbiol. Biotechnol., 34, 373–379 (2007).
2, 115–122 (2007). 33) Heng NCK, Swe PM, Ting YT, Dufour M, Baird HJ, Ragland
7) Lucas R, Grande MA, Abriouel H, Maqueda M, Ben Omar N, NL, Burtenshaw GA, Jack RW, and Tagg JR, Int. Congr. Ser.,
Valdivia E, Martinez-Canamero M, and Galvez A, Food Chem. 1289, 351–354 (2006).
Toxicol., 44, 1774–1781 (2006). 34) Swe PM, Heng NC, Ting YT, Baird HJ, Carne A, Tauch A,
8) Weller DM, Ann. Rev. Phytopathol., 26, 379–407 (1988). Tagg JR, and Jack RW, Microbiology, 153, 3218–3227 (2007).
9) Weller DM, Raaijmakers JM, Gardener BB, and Thomashow 35) van der Merwe IR, Bauer R, Britz TJ, and Dicks LM, Int. J.
LS, Ann. Rev. Phytopathol., 40, 309–348 (2002). Food Microbiol., 92, 153–160 (2004).
10) Kim H, Park J, Choi S, Choi K, Lee G, Ban S, Lee C, and Kim 36) Atanassova M, Choiset Y, Dalgalarrondo M, Chobert JM,
C, J. Microbiol., 41, 196–201 (2003). Dousset X, Ivanova I, and Haertle T, Int. J. Food Microbiol., 87,
11) Risoen PA, Ronning P, Hegna IK, and Kolsto AB, J. Appl. 63–73 (2003).
Microbiol., 96, 648–655 (2004). 37) Lavermicocca P, Valerio F, Evidente A, Lazzaroni S, Corsetti
12) Le Marrec C, Hyronimus B, Bressollier P, Verneuil B, and A, and Gobbetti M, Appl. Environ. Microbiol., 66, 4084–4090
Urdaci MC, Appl. Environ. Microbiol., 66, 5213–5220 (2000). (2000).
13) Kamoun F, Mejdoub H, Aouissaoui H, Reinbolt J, Hammami A, 38) Delves-Broughton J, Food Technol., 4,100–117 (1990).
and Jaoua S, J. Appl. Microbiol., 98, 881–888 (2005). 39) Sebei S, Zendo T, Boudabous A, Nakayama J, and Sonomoto K,
14) Cherif A, Rezgui W, Raddadi N, Daffonchio D, and Boudabous J. Appl. Microbiol., 103, 1621–1631 (2007).
A, Microbiol. Res., 163, 684–692 (2008). 40) Faheem F, Saeed S, and Rasool SA, J. Bot., 39, 1293–1302
15) Jung WJ, Mabood F, Souleimanov A, and Smith DL, Microbiol. (2007).
Res., 167, 14–19 (2011). 41) Lee KD, Gray EJ, Mabood F, Jung WJ, Charles T, Clark SR, Ly
16) Walker JE and Abraham EP, Biochem. J., 118, 557–561 A, Souleimanov A, Zhou X, and Smith DL, Planta, 229, 747–
(1970). 755 (2009).
17) Shelburne CE, An FY, Dholpe V, Ramamoorthy A, Lopatin DE, 42) Gray EJ, Lee KD, Souleimanov AM, di Falco MR, Zhou X, Ly
and Lantz MS, J. Antimicrob. Chemother., 59, 297–300 (2007). A, Charles TC, Driscoll BT, and Smith DL, J. Appl. Microbiol.,
18) Peypoux F, Guinand M, Michel G, Delcambe L, Das BC, and 100, 545–554 (2006).
Lederer E, Biochemistry, 17, 3992–3996 (1978). 43) de Kwaadsteniet M, Todorov SD, Knoetze H, and Dicks LMT,
19) Phae C, Shoda M, Kita N, Nakano M, and Ushiyma K, Ann. Int. J. Food Microbiol., 105, 433–444 (2005).
Phytopatol. Soc. Jpn., 58, 329–339 (1992). 44) Yurong G, Dapeng L, Yan S, and Xiaoyan L, Food Control, 22,
20) Sanders ME, Morelli L, and Tompkins TA, Comp. Rev. Food 657–661 (2011).
Sci. Food Safety, 2, 101–110 (2003). 45) Gupta SK, Mathew KA, Shyam KR, and Sharma A, Plant Dis.
21) Gurtler V and Stanisich VA, Microbiology, 142 (Pt 1), 3–16 Res., 14, 20–24 (1999).
(1996). 46) Pattan HD, Gothkar AK, Joshi P, Chivasa A, and Nyamudeza P,
22) Sambrook J, Fritsch E, and Maniatis T, ‘‘Molecular Cloning, a Agric. Syst., 69, 151–164 (2001).
Laboratory Manual’’ 2nd edn., Cold Spring Harbor Laboratory 47) Portier P, Fischer-Le Saux M, Mougel C, Lerondelle C,
Press, Cold Spring Harbor (1989). Chapulliot D, Thioulouse J, and Nesme X, Appl. Environ.
23) Felsenstein J, Evolution, 39, 783–791 (1985). Microbiol., 72, 7123–7131 (2006).
24) Tagg JR and McGiven AR, Appl. Microbiol., 21, 943–944
(1971).

View publication stats