Available via license: CC BY 4.0
Content may be subject to copyright.
R E S E A R C H Open Access
Characterization of bacteriocin ABC
transporter ATP-binding protein produced
by a newly isolated Enterococcus
casseliflavus MI001 strain
Indira Mikkili
1
, Venkateswarulu TC
1*
, Abraham Peele Karlapudi
1
, Vidya Prabhakar Kodali
2
and Krupanidhi Srirama
1*
Abstract
Background: ATP-binding cassette (ABC) transporters constitute one of the largest transporter protein families and
play a role in diverse biological processes.
Results: In the present study, bacteriocin isolated from the Enterococcus casseliflavus MI001 strain was identified as
an ABC transporter ATP-binding protein. The optimal conditions for the production of bacteriocin were found to be
at 35 °C, a pH 5.5, and an incubation time of 24 h. Purification was performed using ammonium sulphate
precipitation, gel filtration, and DEAE ion exchange chromatography. The bacteriocin was purified with an
eightfold purification scheme resulting with a specific activity of 15,000 AU/mg. The NMR spectrum of purified
bacteriocin revealed the presence of amino acids, namely lysine, methionine, cysteine, proline, threonine,
tryptophan, and histidine. Further, the bacteriocin ABC transporter showed antimicrobial activity against food
spoilage microorganisms.
Conclusions: The ABC transporter ATP-binding protein could be used as a potential alternative for food
preservation, and it may be considered as a bio-preservative agent in food processing industries.
Keywords: Bacteriocin, Enterococcus casseliflavus MI001 strain, ABC transporter, Three-step purification, NMR
spectrum
1 Introduction
Living organisms depend on various means of trans-
port for the uptake of external nutrients and seques-
tration of waste products into the surrounding
environment [3]. ATP-binding cassette (ABC) trans-
porters are one of the largest super families of trans-
port proteins present in all forms of life. They play
diverse roles in both prokaryotes and eukaryotes.
These transporters are the primary transporters func-
tioning as both importers and exporters. Importers
mediate the uptake of essential nutrients, vitamins,
and trace metals from the surrounding environment,
whereas exporters export substrates to the surround-
ing environment. Based on the type of substrate
exported, they are categorized into various types. One
such substrate is bacteriocin, and the transporter pro-
teins exporting bacteriocin are called bacteriocin ABC
transporters. They export bacteriocin across the cell
membrane via a proteolytic function. The proteolytic
domain resides in the N-terminal region, the ABC
transporter domain is in the C-terminal and central
multi-pass transmembrane region. Bacteriocins syn-
thesized as propeptides are processed through this
ABC transporter system and exported from cells as
mature peptides [7]. Bacteriocins are secreted by ei-
ther a double glycine leader in the N-terminal part of
the pre-bacteriocin or secreted by a sec-system [12].
Due to the presence of a glycine leader on the N-
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made.
* Correspondence: venki_biotech327@yahoo.com;
krupanidhijuly2012@gmail.com
1
Department of Bio-Technology, Vignan’s Foundation for Science,
Technology & Research, Vadlamudi, Andhra Pradesh 522213, India
Full list of author information is available at the end of the article
Beni-Suef University Journal o
f
Basic and Applied Sciences
Mikkili et al. Beni-Suef University Journal of Basic and Applied Sciences
(2019) 8:5
https://doi.org/10.1186/s43088-019-0006-z
terminal side, these transporters remove the N-ter-
minal leader peptide from its bacteriocin precursor by
cleavage at a Gly-Gly bond and transport the mature
bacteriocin across the cytoplasmic membrane. The
transporter acts as an efflux system, which helps the
secretion of bacteriocins, proteins, polysaccharides,
toxic compounds, and enzymes. The importance of
these transporters in the multiple cellular functions
and biosynthetic pathways of bacteria represents a
novel strategy for the secretion of bacterial proteins
andpointsoutpotentialdrugtargets[10]. ABC trans-
porters were found to play a key role in virulence
and also identified as suitable targets for the develop-
ment of antibacterial vaccines [1,4]. However, an im-
proved understanding of the structure, function, and
action mechanism of these transporters have enabled
us to develop new approaches for investigating lead
molecules in terms of treating various diseases caused
by problematic organisms [8]. An attempt has been
made to characterize the bacteriocin ABC transporter
ATP-binding protein from the Enterococcus casselifla-
vus MI001 strain.
2 Methods
2.1 Bacterial strains and culture medium
The indicator organisms were procured from the MTCC
at the Institute of Microbial Technology, Chandigarh.
The cultures were revived and stored in refrigerated
conditions for further work. A nutrient broth medium
(Hi-Media Chemicals, India) was used for maintenance
and growing the indicator organisms for bacteriocin ac-
tivity. The de Man, Rogosa, and Sharpe (MRS) broth
medium (Hi-Media Chemicals, India) was used for pro-
duction of the bacteriocin. Finally, the Muller–Hinton
agar (Hi-Media Chemicals, India) medium was used for
bacteriocin activity.
2.2 Optimization of culture conditions for bacteriocin
production
2.2.1 Effect of incubation temperature and pH on
bacteriocin production
Optimum conditions were determined by growing the
test organism at a wide range of temperatures and pH
values. The MRS broth was inoculated with E. casselifla-
vus MI001 and incubated at different temperatures
A
(a) (b) (c)
Fig. 1 AEffect of temperature on bacteriocin production after 24 h against (a) P. aeruginosa, (b) S. aureus, and (c) E.coli
A
(a) (b) (c)
Fig. 2 AEffect of pH on bacteriocin production after 24 h against (a) P. aeruginosa, (b) S. aureus, and (c) E.coli at 37 °C
Mikkili et al. Beni-Suef University Journal of Basic and Applied Sciences (2019) 8:5 Page 2 of 7
(25 °C, 30 °C, 35 °C, 40 °C, and 45 °C) and pH values ran-
ging from 5 to 9 for 48 h. The samples were centrifuged
at 10,000 rpm for 15 min. The supernatant collected was
assayed for bacteriocin activity via the agar well diffusion
method [15,16].
2.2.2 Growth curve and production of bacteriocin
For growth curve measurement and production of
bacteriocin, MRS broth (dextrose—20 g, peptone—10
g, beef extract—8 g, yeast extract—4g, dipotassium
phosphate—2 g, triammonium citrate—2 g, sodium
acetate—5 g, magnesium sulphate—0.2 g, manganese
sulphate—0.05 g, Tween 80—1 g, distilled water—1000
ml) with a pH of 6.5–7.0 was inoculated with 1% v/v
of overnight-grown E. casseliflavus MI001, then incu-
bated at 35 °C for 36 h. For every 3-h time interval,
the optical density at 600 nm and pH were deter-
mined. Bacteriocin activity was determined every 3 h
by agar well diffusion assay. Bacteriocin activity was
measured using the formula AU/mL = 1000/V×D
(where AU stands for arbitrary unit, Vis the volume
of the cell-free supernatant, and Dis the dilution fac-
tor). Specific activity was measured as bacteriocin ac-
tivity (AU)/total protein (mg) [17].
2.2.3 Purification of bacteriocin
Cell-free supernatant was saturated using 70% ammo-
nium sulphate at ice-cold conditions (4 °C) with con-
tinuous stirring (300 rpm) and incubated overnight to
precipitate the proteins. The precipitate was centri-
fuged at 6000 rpm for 10 min and dialyzed overnight
at 4 °C using a 5 mM Tris buffer (pH 7.0). The dialy-
sis sample was subjected to gel filtration chromatog-
raphy using Sephadex G25 (13 mm radius × 150 mm
length) pre-equilibrated with 100 mM phosphate buf-
fer (pH 7.0), and the flow rate was set to 1 ml/5 min
using the same buffer. The active fractions collected
from the gel filtration chromatography were subjected
Fig. 3 Growth curve and bacteriocin production by Enterococcus casseliflavus MI001 strain
Table 1 Summary of purification profile of bacteriocin ABC transporter ATP-binding protein produced by Enterococcus casseliflavus
MI001 strain
Type of sample Volume collected (ml) Total protein (mg) Total activity (AU) Specific activity (AU/mg) Fold purification
Crude 150 129 240,000 1860 1
Ammonium sulphate precipitation
and dialysis
10 3.6 18,000 5000 2.688
Gel filtration chromatography
(SephadexG-75)
5 1.2 11,000 9166 4.927
Ion exchange chromatography
(DEAE cellulose)
2 0.32 4800 15,000 8.064
Mikkili et al. Beni-Suef University Journal of Basic and Applied Sciences (2019) 8:5 Page 3 of 7
toionexchangechromatographyusingaDEAEcellu-
lose column (13 mm radius × 150 mm length) equili-
brated with 25 mM Tris-HCl and 25–150 mM NaCl
buffers (pH 7.0) [16]. The protein concentration was
determined for all fractions, and an antimicrobial
assay was performed against Staphylococcus aureus.
2.2.4 FTIR spectroscopy and NMR analysis
The purified fraction collected was subjected to FTIR ana-
lysis, and the spectrum was recorded in the frequency
range of 4000 to 400 cm
−1
using Agilent Technologies.
The NMR spectrum was recorded for a purified sample
using a Bruker 400-MHz spectrophotometer at 295 K.
The purified bacteriocin sample was dissolved in methanol
and analysed for
1
Hand
13
C NMR spectra. The chemical
shifts were recorded using tetramethylsilane (TMS) as an
internal reference [9].
3 Results and discussion
3.1 Optimization of culture conditions for bacteriocin
activity
3.1.1 Effect of temperature
Temperature influences the growth and production of
bacteriocin, and it is one of the physical factors that
play an important role in the metabolic activities of
bacteria. From Fig. 1A (a, b, c), it was found that the
Fig. 4 Purification of bacteriocin by gel filtration chromatography. The blue line shows the protein content in terms of absorbance values at
280 nm, and the brown line shows the peak pertaining to bacteriocin activity
Fig. 5 FT-IR spectral analysis of purified bacteriocin from Enterococcus casseliflavus MI001 strain
Mikkili et al. Beni-Suef University Journal of Basic and Applied Sciences (2019) 8:5 Page 4 of 7
E. casseliflavus MI001 grown in MRS broth at 35 °C
showed high antimicrobial activity against the indicator
organisms Pseudomonas aeruginosa,S. aureus,and
Escherichia coli after 24 h of incubation, which disap-
peared after 48 h of incubation. After an increase in
temperature up to 40 °C for 24 h of incubation, bacteri-
ocin production was at a minimum, as it showed a re-
duction in antimicrobial activity. Bacteriocin activity
was not observed at temperatures of 25 °C, 30 °C, and
45 °C when incubated for 24 and 48 h. Zotta et al. [18]
also reported that the cultivation of Lactobacillus plan-
tarum at 25 °C reduced the growth compared to the op-
timal temperature of 35 °C. Our results suggest that the
bacterium is surviving in human body conditions; this
feature helps in that this organism could be used as a
probiotic. In consonance, a recent study by Phumisanti-
phong et al. [13] reported that the bacteriocin produc-
tion from Enterococcus faecalis 478 was found to be
high at 37 °C.
3.2 Effect of pH
The synthesis of metabolites and metabolic activities
of bacteria are usually affected by varying the pH. It
was found that E. casseliflavus MI001 incubated for
24 h at pH 5.0–8.0 supported the growth and produc-
tion of bacteriocin with good antimicrobial activity, as
represented in Fig. 2A (a, b, c). The bacteriocin activ-
ity was not observed at acidic pH values and above a
pH of 9.0. This result suggests that the bacteriocin
activity is not due to organic acid production by
bacteria. Iyapparaj et al. [6]reportedthathighbac-
teriocin production was recorded above a pH of 5.0,
and reduced activity was observed at a pH of 9.0.
3.3 Growth curve and production of bacteriocin
Growth of the bacterium under various physical con-
ditions, along with medium components, is import-
ant for the production of bacteriocin. MRS is a
complex synthetic medium which supports bacteri-
ocin production. Higher bacteriocin production was
observed at a temperature of 35 °C, a pH of 5.5, and
27 h of incubation time, as represented in Fig. 3.
Bacteriocin production was high during the exponen-
tial phase and was found to be 1500 AU/ml. A re-
duction in bacteriocin production was observed at
temperatures above 35 °C, pH values below 4.5, and
incubation times in excess of 30 h. In a previous re-
port by Todorov et al. [17], it was found that the
production of bacteriocin was high at 37 °C and pH
5.5. According to Fabricio et al. [2], bacteriocin pro-
duction was high at 37 °C, pH 5.0, and an incubation
time of 8 h during the exponential growth phase.
3.4 Purification of bacteriocin
The crude extract produced by Enterococcus casseliflavus
MI001 was purified by a three-step procedure, as repre-
sented in Table 1. The crude extract precipitated with
70% ammonium sulphate increased the specific activity
about 2.6-fold. The active sample purified by gel filtra-
tion chromatography showed two peaks, the specific ac-
tivity increased about 4.9-fold, and the fractions from 24
to 28 showed high antimicrobial activity against the S.
aureus (15 mm) (Fig. 4). In anion exchange chromatog-
raphy, an 8.0-fold increase in specific activity at 15,000
AU/mg was observed. During each purification step, a
considerable loss in protein concentration and a marked
increase in the specific activity of the bacteriocin were
observed. The purified fraction containing bacteriocin
revealed the molecular weight of a 22-kDa protein via
Table 2 Wave number represents the presence of bonds
Wave number cm
−1
Possible bonds Intensity
3367.186 Amine stretch (N–H) Medium
1635.441 Alkene (C=C) Variable
1096.238 C–N Medium, weak
Table 3
13
C NMR spectral data. Various groups of carbon being detected in purified bacteriocin ABC transporter isolated from E.
casseliflavus MI001 strain
13
C NMR spectra standard values Purified bacteriocin sample NMR spectra (predicted values)
Carbon environment Chemical shift (ppm) Chemical shift (ppm)
C=O (in ketones) 205–220 217.51, 207.26
C in aromatic rings 125–150 146.65
C=C (in alkenes) 115–140 115.46, 114.12, 112.90
C=C (in alkynes) 90–100 96.57
RCH
2
O−50–90 80.02, 61.16, 58.24
RCH
2
Cl 30–60 49.86, 49.64, 49.43, 49.21, 49.01, 48.79, 48.57
RCH
2
NH
2
30–65
Mikkili et al. Beni-Suef University Journal of Basic and Applied Sciences (2019) 8:5 Page 5 of 7
SDS-PAGE [5]. In a recent study reported by Qianwen
et al. [14], it was found that the bacteriocin produced by
Enterococcus faecalis TG2 has a molecular weight of
6.33 kDa protein using a three-step purification
procedure.
3.5 FTIR spectrum and NMR spectral analysis
The FTIR spectral analysis showed the presence of a
C=C stretch and amine group coupled with a C–N
group (Fig. 5). The correlation of various peaks is
represented in Table 2. A previous study by Kadir-
velu et al. [9] reported the presence of an amide and
hydroxyl group coupled with C–H. Further,
13
C
NMR and
1
H NMR analyses were carried out to dis-
cover the different amino acids and carbon com-
pounds forming the molecular structure of the
bacteriocin of Enterococcus casseliflavus MI001. The
NMR spectra obtained were compared with the
standard
13
C NMR spectrum presented in Table 3.
From Fig. 6, it can be seen that the
13
CNMR
showed the presence of 17 carbons, of which 2 are
in C=O (ketones), 1 is in an aromatic ring, 3 are in
C=C (alkenes), 1 is in C=C (alkyne), 3 are in R-
CH
2
O, and 7 are in the carbon environment R-
CH
2
Cl and RCH
2
NH
2
.The
1
H NMR spectrum was
compared with the peaks of a standard spectrum of
20 amino acids. Of the 20 amino acids, seven, viz.,
lysine, methionine, cysteine, proline, threonine, tryp-
tophan, and histidine, were involved in forming the
peptide structure of the bacteriocin of E. casselifla-
vus MI001, as represented in both Fig. 6b(Right)
and Table 4. In a previous study by Neha et al. [11],
it was reported that the purified bacteriocin of
Lactobacillus brevis UN examined by a
1
HNMR
spectrum exhibited the following amino acids: pro-
line, glutamic acid, arginine, leucine, isoleucine, and
serine.
4 Conclusions
In this study, the optimized conditions for the bac-
teriocin production and three-step procedure for
purification yielded a bacteriocin peptide molecule
with antimicrobial activity. The purified bacteriocin
molecular weight was found to be 22 kDa, which
matched the results from MALDI-TOF. The
13
C
NMR and
1
H NMR analyses revealed that the carbon
environment and combinations of the unique amino
acids in purified bacteriocin result in antibacterial
activity, which has been reported for the first time
through the present study. The antimicrobial activity
of this ABC transporter bacteriocin has attracted
much attention due to the antimicrobial resistance
of the bacteria, which can be used as a next-gener-
ation antimicrobial in various fields.
Fig. 6
13
C NMR spectra (Left) and
1
H spectra (Right) of purified bacteriocin isolated from E. casseliflavus MI001 strain
Table 4
1
H NMR spectral data, delta (ppm) of various amino acids detected in purified bacteriocin of E. casseliflavus MI001
Name of the amino acid Chemical shift (ppm)—standard values Chemical shift (ppm)—predicted values
Lysine 1.7 1.713
Methionine 2.0 2.065
Cysteine 3.2 3.270
Proline 2.1–3.6 3.320–3.391
Threonine 4–5 4.866
Tryptophan 6.5–7.8 7.332
Histidine 6.5–8.5 8.433
Mikkili et al. Beni-Suef University Journal of Basic and Applied Sciences (2019) 8:5 Page 6 of 7
Abbreviations
ABC: ATP-binding cassette; MTCC: Microbial Type Culture Collection;
NMR: Nuclear magnetic resonance
Acknowledgements
The authors would like to acknowledge the facilities supported by DST-FIST
and Vignan’s Foundation for Science Technology and Research (Deemed to
be University), Guntur, India.
Authors’contributions
MI carried out the experimental studies and drafted the manuscript. SK and
KV participated in the design of the study. KA and TCV participated in
optimization studies. KA and TCV conceived of the study, participated in its
design and coordination, and helped to draft the manuscript. All authors
read and approved the final manuscript.
Funding
Not applicable.
Availability of data and materials
All data generated or analysed during this study are included in this
published article.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Bio-Technology, Vignan’s Foundation for Science,
Technology & Research, Vadlamudi, Andhra Pradesh 522213, India.
2
Department of Bio-Technology, Vikrama Simhapuri University, Nellore,
Andhra Pradesh 524001, India.
Received: 5 July 2019 Accepted: 26 July 2019
References
1. Davidson AL, Elie D, Cedric O, Jue C (2008) Structure, function, and evolution
of bacterial ATP-binding cassette systems. Microbiol Mol Biol Rev 72:317–364
2. Fabricio LT, Bruna CG, Elaine CPDM (2011) Partial purification and
characterization of a bacteriocin produced by Enterococcus faecium 130
isolated from mozzarella cheese. Ciencia e Tecnologia de Alimentos 31:
155–159
3. Gizaw M, Anandakumar P (2017) A review on ATP binding cassette (ABC)
transporters. Int J Pharma Res Health Sci 5:1607–1615
4. Helen SG, Richard WT (2004) ATP-binding cassette transporters are targets
for the development of antibacterial vaccines and therapies. Infect Immun
72:6757–6763
5. Indira M, Venkateswarulu TC, Vidya Prabhakar K, Abraham Peele K,
Krupanidhi S (2018) Isolation and characterization of bacteriocin producing
Enterococcus casseliflavus and its antagonistic effect on Pseudomonas
aeruginosa. Karbala Int J Modern Sci 4:361–368
6. Iyapparaj P, Thirumalai M, Ramasamy R, Santhiyagu P, Chandrasekaran K,
Grasian I, Arunachalam P (2013) Optimization of bacteriocin production by
Lactobacillus sp. MSU3IR against shrimp bacterial pathogens. Aquatic
Biosystems 9:12
7. Jan M, Gunter D, Jos V, Chuanwu X (2001) Processing and export of peptide
pheromones and bacteriocins in Gram negative bacteria. Trends Microbiol
9(4):164–168
8. Joni T, Glenn ST (2010) ABC transporters in microorganisms. Expert Rev
Anti-Infect Ther 8(4):375–377
9. Kadirvelu J, Venkatasubramanian V, Perumal JA, Appukuttan S,
Muthukandan U (2015) Characterization of an antibacterial compound,
2-hydroxyl indole-3-propanamide, produced by lactic acid bacteria
isolated from fermented batter. Appl Biochem Biotechnol 177:137–147
10. Murphy TF, Brauer AL, Johnson AKC (2016) ATP-binding cassette (ABC)
transporters of the human respiratory tract pathogen, Moraxella catarrhalis:
role in virulence. PLoS One 11(7):e0158689
11. Neha G, Nivedita S, Ahlawat OP (2014) Purification and characterization
of bacteriocin produced by Lactobacillus brevis UN isolated from
Dhulliachar: a traditional food product of north East India. Indian J
Microbiol 54(2):185–189
12. Ness IF, Dzung BD, Yasuyoshi I (2014) Enterococcal bacteriocins and
antimicrobial proteins that contribute to niche control. In: Gilmore MS,
Clewell DB, Ike Y et al (eds) Enterococci: from commensals to leading
causes of drug resistant infection. Massachusetts Eye and Ear Infirmary,
Boston
13. Phumisantiphong U, Siripanichgon K, Reamtong O, Diraphat P (2017) A
novel bacteriocin from Enterococcus faecalis 478 exhibits a potent
activity against vancomycin-resistant enterococci. PLoS One 12(10):
e0186415
14. QianwenX,JinW,RenpengD,FangkunZ,HanY,ZhijiangZ(2018)
Purification and characterization of bacteriocin produced by a strain
of Enterococcus faecalis TG2. Appl Biochem Biotechnol 184:1106–1119
15. Sivaramasamy E, Neelamegam A, Packiyam M, Thangavel B (2014)
Production, purification and characterization of bacteriocin from
Lactobacillus murinus AU06 and its broad antibacterial spectrum. Asian Pac J
Trop Biomed 4(11):S305–S311
16. Sure KP, Kotnis PV, Bhagwat PK, Ranveer RC, Dandge PB, Sahoo AK
(2016) Production and characterization of bacteriocin produced by
Lactobacillus viridescence (NICM 2167). Braz Arch Biol Technol 59:
e16150518
17. Todorov SD, Manuela V, Bernadette DGF, Wilhelm HH (2013) Partial
characterization of bacteriocins produced by three strains of Lactobacillus
sakei, isolated from salpicao, a fermented meat product from North-West of
Portugal. Food Control 30:111–121
18. Zotta T, Guidone A, Ianniello RG, Parente E, Ricciardi A (2013) Temperature
and respiration affect the growth and stress resistance of Lactobacillus
plantarum C17. J Appl Microbiol 115(3):848–858
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Mikkili et al. Beni-Suef University Journal of Basic and Applied Sciences (2019) 8:5 Page 7 of 7
