INTRODUCTION
Pathogenic microorganisms resistant to traditional
antibiotics are a serious problem of modern healthcare.
There is evidence that over 70% of all pathogenic
bacteria are resistant to at least one of the most
commonly used antibiotics. Therefore, there is an urgent
need for new drugs and therapeutic approaches to
overcome their resistance [1–5].
Antimicrobial peptides produced by various
organisms from bacteria to mammals are an ideal
alternative to antibiotics due to their antimicrobial, antiinflammatory,
angiogenic, and immunomodulatory
properties, as well as low bacterial resistance [6].
However, their use is limited by toxicity and stability
in vivo [7].
Antimicrobial peptides act against various types of
pathogens, including gram-positive and gram-negative
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bacteria, viruses, and microscopic fungi, through the
destruction of the cytoplasmic membrane, intracellular
penetration, and immunomodulation [8, 9]. Structurally,
antimicrobial peptides are classified into linear cationic
amphipathic peptides and macrocyclic peptides [10].
As a rule, antimicrobial peptides are short peptides
consisting of 10–50 amino acids [11, 12]. They have
common features despite differing in length, amino acid
sequences, and conformation [13]. Typical antimicrobial
peptides are composed of positively charged residues
such as arginine, lysine, and histidine [14]. Cationic
peptides with a positive charge ranging from +2 to +11
can interact with the membranes of microbial cells.
Besides, a significant part of antimicrobial peptides
is hydrophobic, contributing to the formation of
amphipathic secondary or quaternary structures [15].
Antimicrobial peptides have several advantages
over traditional antibiotics [16]. First of all, they have
a broad spectrum of antimicrobial activity, against
even multidrug-resistant pathogens [8, 16]. Secondly,
antimicrobial peptides are highly active against gramnegative
bacteria, which are more serious targets than
gram-positive bacteria [17]. Another advantage is a
rather low likelihood of drug resistance.
Bacteriocins are antimicrobial, ribosomally
synthesized peptides of bacteria with a low molecular
weight [18]. Mostly studied are bacteriocins produced
by lactobacilli. They can be roughly divided into four
categories: lantibiotics (e.g., nisin); non-antibiotic
bacteriocins with good activity against Listeria
monocytogenes, as well as pediocins, which make up
the largest group; thermosensitive macromolecular
proteinaceous bacteriocins; and complex bacteriocins
with carbohydrates, lipids, and proteins [19–23]. Of all
well-studied bacteriocins of lactobacilli, only nisin is
produced commercially [24].
Potential sources of bacteria producing bacteriocins
are dairy products, cow rumen, feed, as well as natural
objects such as soils, plant waste, rhizosphere of plants,
bottom sediments of water bodies, etc. [18, 25, 26].
In our previous studies, we isolated 19 microorganisms
from the natural sources of Kemerovo Region
(Siberian Federal District, Russia), including 10 species
of bacteria (Geobacillus, Bacillus, Lactobacillus,
Leuconostoc, and Pediococcus) that showed high
antimicrobial activity against Escherichia coli, Salmonella
enterica, Staphylococcus aureus, Pseudomonas
aeruginosa, Bacillus mycoides, Candida albicans, and
Penicíllium citrinum [27–29].
In this study, we aimed to examine the structure
and properties of antimicrobial peptides produced by
antagonist microorganisms isolated from the natural
objects in Siberia.
STUDY OBJECTS AND METHODS
Our study objects were bacteria isolated from the
natural sources of Kuzbass (Table 1).
Microorganism cultures. To obtain enrichment
cultures of microorganisms, we crushed the samples
of soil, bottom sediments, and plant waste under
sterile conditions and rubbed their small amounts on
Petri dishes with nutrient agar. The Petri dishes were
incubated for three days at 26°C. Two nutrient media
were used: lactobacilli were cultured on MRS agar;
Bacillus and Geobacillus bacteria were cultured on
a medium (pH 7.4 ± 0.2) containing 10.0 g/L casein
hydrolysate, 2.5 g/L yeast extract, 5.0 g/L glucose,
2.5 g/L potassium hydrogen phosphate, and 12.0 g/L
bacteriological agar.
Pure cultures of microorganisms were obtained
from enrichment cultures by streaking. Microorganisms
were cultivated on the media described above for 24 h:
Lactobacillus, Leuconostoc and Pediococcus bacteria at
37°C, and Bacillus and Geobacillus at 30°C.
At the end of cultivation, cell debris was removed
from all suspension cultures. The cultures were
centrifuged at 3900 rpm in plastic flasks. The resulting
supernatant was dried in a Labcocnco Triad freeze
dryer (Labcocnco, USA) at a freezer temperature of
–80°С, supernatant temperature of –20°С, and 0.05 mbar
vacuum.
Protein fractions. To separate protein into
individual fractions, the dried biomass was dissolved in
1 mL of 0.25 M phosphate buffer and the total protein
was precipitated by adding 2 mL of concentrated
Table 1 Study objects
Microorganism Reference Source of isolation
Bacillus subtilis Bs-1 Soil (Peshcherka village, Kemerovo district)
Lactobacillus plantarum Lp-7 Rhizosphere of plants (Voznesenka village, Yaya district)
Leuconostoc mesenteroides Lm-8
Pediococcus acidilactici Pa-9 Rhizosphere of plants (Ursk village, Guryevsk district)
Pediococcus pentosaceus Pp-11 Plant waste at Sukhovsky farm (Kemerovo city)
Lactobacillus casei Lc-12
Lactobacillus fermentum Lf-13 Plant waste at Niva farm (Gorskino village, Guryevsk district)
Pediococcus damnosus Pd-16 Plant waste at Veles farm (Yaya village, Yaya district)
Geobacillus stearothermophilus Bs-19 Bottom sediments of the Kara-Chumysh reservoir (Prokopyevsk district)
Bacillus caldotenax Bc-20 Bottom sediments of Lake Udai (Mariinsk district)
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ammonium sulfate solution. The resulting protein
suspension was separated by centrifugation at 8000 rpm.
The protein precipitate was dissolved in 1 mL of
0.025 M Tris buffer solution (pH 4.5). The precipitate
was applied to an Enrich 650 10 mm X 300 mm column
(Biorad, USA) for a gel permeation high performance
liquid chromatography (HPLC) at 280 nm using a direct
injection system. Fractionation was performed using an
NGC fraction collector (Biorad, USA).
Additionally, each protein fraction was purified
on hydrophobic Amberlite XAD X-6 resins by
chromatography. A glass column was filled with 10 g
of Amberlite XAD-2 resin equilibrated with 10 mL of
20 mM trifluoroacetic acid solution. A protein solution
in an acetate buffer was applied to the column and
eluted in a methanol gradient from 0 to 15%, with a
gradient rise of 5% for every 10 fractions. Fractions
containing proteins were determined by taking 50 μL
of each fraction and mixing it with a solution of
Bradford’s reagent in a 1:1 ratio. The resulting
solution was measured on a Biorad SmartSpec Plus
Spectrophotometer (USA). Fractions with an optical
absorption of 0.06 or more were selected for further
drying and identifying the amino acid sequence by
the MALDI-TOF method using a MALDI TOF/TOF
BRUKER Autoflex Speed mass spectrometer (Bruker
Corporation, USA)
Trypsinolysis. Peptides were precipitated by adding
an equal volume of methanol/chloroform mixture to an
aliquot of a 200 μL fraction. The resulting precipitate
was separated by centrifugation at 4000 rpm. The
precipitate was dissolved in 100 μL of 6 M urea solution,
to which 5 μL of dithiothreitol (DTT) solution was
added to keep for 60 min at room temperature. Then,
we added 20 μL of iodoacetamide solution and kept the
mixture for 60 min at room temperature. After that, we
added 20 μL of a DTT solution and kept the mixture
again for 60 min at room temperature. After adding
775 μL of MiliQ H2O and 50 μL of trypsin solution,
the mixture was stirred by pipetting and kept in a
thermostat at 37°C for 12 h. The enzyme was inactivated
by adding 10 μL of trifluoroacetic acid. The peptides
were purified by chromatography on C18 cartridges.
The reaction mixture was applied to a cartridge and
eluted with a solution of 0.1% trifluoroacetic acid in a
1:1 H2O/acetonitrile mixture. Analysis and Top-Dawn
sequencing were performed on 1 μL of a purified peptide
solution.
The antibacterial properties of the peptides
against Bacillus pumilus and Escherichia coli were
measured by the disk diffusion method. For this, we
used suspensions of night cultures grown on a standard
liquid nutrient LB medium with a titer of 0.5. The
number of microorganisms (titer) in the suspension was
determined by optical density at 595 nm. 200 μL of
the pathogen culture was dropped onto a 90 mm Petri
dish, rubbed with a sterile spatula by the spread plate
method, and left to dry for 20 min under a laminar
with the lid ajar. Then, 0.5 cm sterile filter disks soaked
in the peptide solutions under study and dried at room
temperature for 10 min were placed on the Petri dishes
in the radial direction. The Petri dishes were left for
30 min at room temperature and then incubated in
a thermostat at 37°C for 12 h. Then, we identified a
bacterial inhibition zone around the disc and measured
its diameter with a vernier caliper. Ampicillin at a
concentration of 5 mg/mL was used as a positive control,
and a disc soaked in a liquid medium was used as a
negative control.
The fungicidal activity of the peptides against
the microscopic fungi Aspergillus flavus and
Aspergillus niger was measured by the disk diffusion
method. The fungi were cultivated for 7 days, with
an inoculation density of 6×107 conidia per 1 mL of
medium. The results were analyzed with time intervals
(3, 9, 12, 24, 48, 72 h, etc.) and by the fungus growth
phase (stationary, accelerated growth, logarithmic),
i.e., during the periods of exponential cell growth,
decreased growth, and death or autolysis. At the end of
the incubation, the inhibition zone around the disc was
measured with a vernier caliper (mm), which indicated
the degree of biocidal activity or its absence. A negative
control was the samples with filters impregnated
with the medium, and a positive control was the
pharmaceutical preparation Irunin® (Veropharm,
Russia) with itraconazole as an active ingredient.
Statistical data were analyzed in Microsoft Office
Excel 2007. All the experiments were carried out in
triplicate. Statistical analysis was performed using
a one-sample Student’s t-test. The differences were
considered statistically significant at P < 0.05.
Table 2 Peptides from the biomass of bacteria isolated from
natural sources of Kuzbass
Microorganism Isolated
fractions
Microorganism Isolated
fractions
Bs-1 Bs-1_1 Lc-12 Lc-12_1
Lp-7 Lp-7_1 Lf-13 Lf-13_1
Lf-13_2
Lf-13_3
Lm-8 Lm-8_1 Pd-16 Pd-16_1
Lm-8_1 Pd-16_2
Pd-16_3
Pd-16_4
Pa-9 Pa-9_1 Bs-19 Bs-19_1
Pa-9_2 Bs-19_2
Pp-11 Pp-11_1 Bc-20 Bc-20_1
Pp-11_2
Pp-11_3
Pp-11_4
Pp-11_5
Pp-11_6
Pp-11_7
Pp-11_8
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RESULTS AND DISCUSSION
Several protein fractions were isolated from the
culture fluid of all the studied samples (Table 2).
According to Table 2, one protein fraction was
isolated from the culture fluid of Bacillus subtilis,
Lactobacillus plantarum, Lactobacillus casei, and
Bacillus caldotenax; two protein fractions from
Leuconostoc mesenteroides, Pediococcus acidilactici,
and Geobacillus stearothermophilus; three protein
fractions from Lactobacillus fermentum; four protein
fractions from Pediococcus damnosus; and eight protein
fractions from the Pediococcus pentosaceus culture
fluid.
The results of the MALDI TOF mass spectrometry
of protein fractions are presented in Figures 1–7. We
found some identical mass spectra of protein fractions
synthesized by different bacteria.
Figure 1 Mass spectrum of fraction Bs-1_1
Figure 2 Mass spectrum of fraction Bc-20_1 (Lf-13_1, Lf-13_2, Lf-13_3)
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Having analyzed the mass spectra, we determined
the molecular masses and amino acid sequences of seven
peptides (Table 3).
Table 3 also shows the presence of analogues for the
studied peptides in the PepBank and Uniprot databases.
We established a homology of fractions Pp-11_1, Pp-
11_2, Pp-11_3, Pp-11_4, Pp-11_5, Pp-11_6, Pp-11_7, Pp-
11_8, Lp-7_1, Pd-16_1, Pd-16_2, Pd-16_3, and Pd-16_4
with the cysteine membrane protein Giardia lamblia P15
(Fig. 8), as well as a homology of peptides Pa-9_1 and
Pa-9_2 with the Planctomycetes bacterium I41 peptides
(Fig. 9). The rest of the peptides had no analogues in the
PepBank and Uniprot databases.
Figure 3 Mass spectrum of fraction Bs-19_1 (Lc-12_1)
Figure 4 Mass spectrum of fraction Bs-19_2
The antibacterial properties of the studied peptides
against gram-positive (Bacillus pumilus) and gramnegative
(Escherichia coli) bacteria, as well as their
fungicidal properties against the microscopic fungi
Aspergillus niger and Aspergillus flavus are presented in
Tables 4–5 and Figs. 10–11.
According to Table 4 and Fig. 10, of the seven
peptides under study, only one (Bs-19_2) exhibited
no antagonistic activity against E. coli and B. pumilus
strains. Peptide fraction Pp-11_1 (and peptides with
identical amino acid sequences Pp-11_2, Pp-11_3, Pp-
11_4, Pp-11_5, Pp-11_6, Pp-11_7, Pp-11_8, Pd-16_1,
Pd-16_2, Pd- 16_3, Pd-16_4, and Lp-7_1) showed
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high antagonism against B. pumilus and pronounced
antibacterial activity against E. coli. Peptides Bs-1_1
and Bc-20_1 (identical Lf-13_1, Lf-13_2, and Lf-13_3),
Lm-8_1 (identical Lm-8_2), and Pa-9_1 (identical
Figure 5 Mass spectrum of fraction Pp-11_1 (Pp-11_2, Pp-11_3, Pp-11_4, Pp-11_5, Pp-11_6, Pp-11_7, Pp-11_8, Lp-7_1, Pd-16_1,
Pd-16_2, Pd-16_3, Pd-16_4)
Figure 6 Mass spectrum of fraction Lm-8_1 (Lm-8_2)
Pa-9_2) had moderate and pronounced antagonistic
activity against B. pumilus, but no activity against
E. coli. Finally, peptide Bs-19_1 (identical Lc-12_1)
showed bacteriostatic activity only against E. coli.
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Figure 7 Mass spectrum of fraction Pa-9_1 (Pa-9_2)
Table 3 Molecular masses and amino acid sequences of peptides from the culture fluid of bacteria isolated from the natural sources
of Kuzbass
Code
of peptide
Molecular
mass of
peptide, Da
Amino acid sequence Analogues
in PepBank
or Uniprot
Bs-1_1 13140.97 AFGKHVLIPVSCGFTYVWKCTLIPHISARPHYCFH
RQHCDYKINQVSFEDAWHTPC
No analogues
Bc-20_1
Lf-13_1
Lf-13_2
Lf-13_3
6577.63 FLAFAYLPIPGWHPDYNGRAMKWANRPFTYICHGR
DLKLRQMLYSGATIGHAEMR
No analogues
Bs-19_1
Lc-12_1
6572.00 PHQGHAFNFSCDMETAGFKGTQFWTFKSV
SPHLATFKLGHMSTYAILGFAGCH
No analogues
Bs-19_2 6290.80 FVKGFHPSMTARGVVSDEADGRCDRFV
KGFHPSMTARGVVSDEADGRCDR
No analogues
Pp-11_1
Pp-11_2
Pp-11_3
Pp-11_4
Pp-11_5
Pp-11_6
Pp-11_7
Pp-11_8
Lp-7_1
Pd-16_1
Pd-16_2
Pd-16_3
Pd-16_4
2061.66 VMCLARKCSQGLIVKAPLM High homology
with cysteine
membrane
protein Giardia
lamblia P15
Lm-8_1
Lm-8_2
35571.18 MOPRKLCQSP VAILKMCVPA RQKVPSILKM OPRKLCQSPV AILKMCVPAR
QKVPSILKMO PRKLCQSPVAILKMCVPARQ KVPSILKMOP RKLCQSPVAI
LKMCVPARQK VPSILKMOPR KLCQSPVAIL KMCVPARQKV PSILKMOPRK
LCQSPVAILK MCVPARQKVP SILKMOPRKL CQSPVAILKM CVPARQKVPS
ILKMOPRKLC QSPVAILKMC VPARQKVPSILKMOPRKLCQ SPVAILKMCV
PARQKVPSIL KMOPRKLCQS PVAILKMCVP ARQKVPSILK MOPRKLCQSP
VAILKMCVPA RQKVPSILK
No analogues
Pa-9_1
Pa-9_2
2587.21 AVPSMKLCIQWSPVRASPCVMLGI High degree of
homology with
Planctomycetes
bacterium I41
peptides
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Figure 9 The closest analogues for peptides Pa-9_1 and Pa-9_2 according to BLAST NCBI
Figure 8 The closest analogues for peptides Pp-11_1, Pp-11_2, Pp-11_3, Pp-11_4, Pp-11_5, Pp-11_6, Pp-11_7, Pp-11_8, Lp-7_1,
Pd-16_1, Pd-16_2, Pd-16_3, and Pd-16_4 according to BLAST NCBI
Table 4 Antibacterial properties of peptides from the culture
fluid of bacteria isolated from the natural sources of Kuzbass
(M ± m)
Peptide code Test strain Lysis zone
diameter, cm
Degree
of activity
Negative
control
Escherichia coli 0 –
Bacillus pumilus 0 –
Ampicillin
(positive
control)
0.5 mg/mL
Escherichia coli 0.90 ± 0.05 –
Bacillus pumilus 2.40 ± 0.10 –
Bs-1_1 Escherichia coli 0 Absent
Bacillus pumilus 0.60 ± 0.03 Moderate
Bc-20_1
Lf-13_1
Lf-13_2
Lf-13_3
Escherichia coli 0 Absent
Bacillus pumilus 0.80 ± 0.04 Pronounced
Bs-19_1
Lc-12_1
Escherichia coli 0.60 ± 0.03 Moderate
Bacillus pumilus 0 Absent
Bs-19_2 Escherichia coli 0 Absent
Bacillus pumilus 0 Absent
Pp-11_1
Pp-11_2
Pp-11_3
Pp-11_4
Pp-11_5
Pp-11_6
Pp-11_7
Pp-11_8
Pd-16_1
Pd-16_2
Pd-16_3
Pd-16_4
Lp-7_1
Escherichia coli 0.70 ± 0.04 Pronounced
Bacillus pumilus 1.00 ± 0.05 High
Lm-8_1
Lm-8_2
Escherichia coli 0 Absent
Bacillus pumilus 0.70 ± 0.04 Pronounced
Pa-9_1
Pa-9_2
Escherichia coli 0 Absent
Bacillus pumilus 0.60 ± 0.03 Moderate
Unlike biocidal properties, which do not depend
on the pathogen growth phase and naturally decrease
over time, fungicidal properties need to be determined
at each stage of the fungus life cycle since fungal
pathogens have a complex growth cycle. We found that
the peptide fractions under study did not stop fungal
growth, but only inhibited it, which was indicated by a
change in the mycelium color. The results were analyzed
with time intervals (3, 9, 12, 24, 48, 72 h, etc.) and by
the fungus growth phase (stationary, accelerated growth,
logarithmic), i.e., during the periods of exponential cell
growth, decreased growth, and death or autolysis. The
samples with filters impregnated with a nutrient medium
were used as a control.
Having analyzed the peptides’ fungicidal activity
(Table 5, Fig. 11), we identified those peptides which
could inhibit Aspergillus growth, rather than stop it
completely. They were Bs-1_1, Bc-20_1 (identical
Lf-13_1, Lf-13_2, and Lf-13_3) and Bs-19_2, with a
lysis zone diameter of 0.1–0.2 mm. The maximum
fungicidal activity against A. niger (0.3–0.5 mm lysis
zone) was demonstrated by peptides Bs-19_1 (identical
Lc-12_1), Pp-11_1 (identical Pp-11_2, Pp-11_3, Pp-11_4,
Pp-11_5, Pp-11_6, p-11_7, Pp-11_8, Pd-16_1, Pd-16_2,
Pd-16_3, Pd-16_4, and Lp-7_1), and Pa-9_1 (Pa-9_2). The
highest activity against A. flavus (0.3–0.4 mm lysis zone)
was revealed by peptides Pp-11_1 (identical Pp-11_2,
Pp-11_3, Pp-11_4, Pp-11_5, Pp-11_6, p-11_7, Pp-11_8,
Pd-16_1, Pd-16_2, Pd-16_3, Pd-16_4, and Lp-7_1), Lm-
8_1 (identical Lm-8_2), and Pa-9_1 (identical Pa- 9_2).
Based on the study of antimicrobial activity, we
selected peptides with maximum antibacterial (against
B. pumilus) and fungicidal (against A. niger and
A. flavus) properties: Pp-11_1 (identical Pp-11_2,
Pp-11_3, Pp-11_4, Pp-11_5, Pp-11_6, p-11_7, Pp-11_8,
Pd-16_1, Pd-16_2, Pd-16_3, Pd-16_4, and Lp-7_1 ), Lm-
8_1 (identical Lm-8_2), and Pa-9_1 (identical Pa-9_2).
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Bs-1_1 Escherichia coli Bs-1_1 Bacillus pumilus Bc-20_1, Lf-13_1, Lf-13_2,
Lf-13_3 Escherichia coli
Bc-20_1, Lf-13_1, Lf-13_2,
Lf-13_3 Bacillus pumilus
Bs-19_1, Lc-12_1
Escherichia coli
Bs-19_1, Lc-12_1
Bacillus pumilus
Bs-19_2 Escherichia coli Bs-19_2 Bacillus pumilus
Pp-11_1, Pp-11_2,
Pp-11_3, Pp-11_4,
Pp-11_5, Pp-11_6,
Pp-11_7, Pp-11_8, Lp-7_1,
Pd-16_1, Pd-16_2,
Pd-16_3, Pd-16_4
Escherichia coli
Pp-11_1, Pp-11_2,
Pp-11_3, Pp-11_4,
Pp-11_5, Pp-11_6,
Pp-11_7, Pp-11_8, Lp-7_1,
Pd-16_1, Pd-16_2,
Pd-16_3, Pd-16_4
Bacillus pumilus
Pa-9_1, Pa-9_2
Escherichia coli
Pa-9_1, Pa-9_2
Bacillus pumilus
Lm-8_1, Lm-8_2
Escherichia coli
Lm-8_1, Lm-8_2
Bacillus pumilus
Amp Escherichia coli Amp Bacillus pumilus
– Escherichia coli – Bacillus pumilus
Figure 10 Antibacterial properties of peptides from
the culture fluid of bacteria isolated from the natural
sources of Kuzbass
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Bs-1_1 Aspergillus niger Bs-1_1 Aspergillus flavus Bc-20_1, Lf-13_1, Lf-13_2,
Lf-13_3 Aspergillus niger
Bc-20_1, Lf-13_1, Lf-13_2,
Lf-13_3 Aspergillus flavus
Bs-19_1, Lc-12_1
Aspergillus niger
Bs-19_1, Lc-12_1
Aspergillus flavus
Bs-19_2 Aspergillus niger Bs-19_2 Aspergillus flavus
Pp-11_1, Pp-11_2,
Pp-11_3, Pp-11_4,
Pp-11_5, Pp-11_6,
Pp-11_7, Pp-11_8, Lp-7_1,
Pd-16_1, Pd-16_2,
Pd-16_3, Pd-16_4
Aspergillus niger
Pp-11_1, Pp-11_2,
Pp-11_3, Pp-11_4,
Pp-11_5, Pp-11_6,
Pp-11_7, Pp-11_8, Lp-7_1,
Pd-16_1, Pd-16_2,
Pd-16_3, Pd-16_4
Aspergillus flavus
Pa-9_1, Pa-9_2
Aspergillus niger
Pa-9_1, Pa-9_2
Aspergillus flavus
Lm-8_1, Lm-8_2
Aspergillus niger
Lm-8_1, Lm-8_2
Aspergillus flavus
Figure 11 Fungicidal properties of peptides from the culture
fluid of bacteria isolated from the natural sources of Kuzbass
Thus, the fact that peptides produced by microorganisms
inhabiting the natural ecosystems of Kuzbass
exhibit antagonistic activity against opportunistic strains
opens up prospects for their use in the production of
pharmaceutical substances with antimicrobial action,
alternative to traditional antibiotics.
CONCLUSION
We identified amino acid sequences and molecular
masses of peptide fractions produced by bacteria
(Lactobacillus, Leuconostoc, Pediococcus, Bacillus,
and Geobacillus) isolated from the natural objects of
the Siberian region (soil, rhizosphere of plants, bottom
sediments of reservoirs, and plant waste). In total,
we isolated 25 protein fractions, some with identical
mass spectra. Thus, we obtained seven peptides with
different amino acid sequences, five of which have
no analogues in the PepBank and Uniprot databases.
One of the peptides (VMCLARKCSQGLIVKAPLM,
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Table 5 Fungicidal properties of peptides from the culture fluid of bacteria isolated from the natural sources of Kuzbass (M ± m)
Peptide code Lysis zone diameter by growth phase, mm
Exponential cell
growth, h
Decreased growth, h Death or autolysis, days
3 9 12 48 72 6 12
Aspergillus niger
Bs-1_1 + + + 0.100 ± 0.005 0.200 ± 0.010 0.100 ± 0.005 0.100 ± 0.005
Bc-20_1, Lf-13_1, Lf-13_2, Lf-13_3 + + + 0.200 ± 0.010 0.100 ± 0.005 0.100 ± 0.005 0.100 ± 0.005
Bs-19_1, Lc-12_1 + + + 0.500 ± 0.025 0.500 ± 0.025 0.400 ± 0.020 0.400 ± 0.020
Bs-19_2 + + + 0.100 ± 0.005 0.100 ± 0.005 0.200 ± 0.010 0.200 ± 0.010
Pp-11_1, Pp-11_2, Pp-11_3, Pp-11_4, Pp-
11_5, Pp-11_6, p-11_7, Pp-11_8, Pd-16_1,
Pd-16_2, Pd-16_3, Pd-16_4, Lp-7_1
+ + + 0.100 ± 0.005 0.200 ± 0.010 0.400 ± 0.020 0.400 ± 0.020
Lm-8_1, Lm-8_2 + + + 0.100 ± 0.005 0.100 ± 0.005 0.100 ± 0.005 0.100 ± 0.005
Pa-9_1, Pa-9_2 + + + 0.100 ± 0.005 0.300 ± 0.015 0.400 ± 0.020 0.400 ± 0.020
Aspergillus flavus
Bs-1_1 + + + 0.100 ± 0.005 0.100 ± 0.005 0.100 ± 0.005 0.100 ± 0.005
Bc-20_1, Lf-13_1, Lf-13_2, Lf-13_3 + + + 0.200 ± 0.010 0.100 ± 0.005 0.100 ± 0.005 0.100 ± 0.005
Bs-19_1, Lc-12_1 + + + 0.100 ± 0.005 0.100 ± 0.005 0.100 ± 0.005 0.100 ± 0.005
Bs-19_2 + + + 0.100 ± 0.005 0.100 ± 0.005 0.100 ± 0.005 0.100 ± 0.005
Pp-11_1, Pp-11_2, Pp-11_3, Pp-11_4, Pp-
11_5, Pp-11_6, p-11_7, Pp-11_8, Pd-16_1,
Pd-16_2, Pd-16_3, Pd-16_4, Lp-7_1
+ + + 0.100 ± 0.005 0.100 ± 0.005 0.300 ± 0.015 0.400 ± 0.020
Lm-8_1, Lm-8_2 + + + 0.100 ± 0.005 0.300 ± 0.015 0.300 ± 0.015 0.400 ± 0.020
Pa-9_1, Pa-9_2 + + + 0.100 ± 0.005 0.300 ± 0.015 0.400 ± 0.020 0.400 ± 0.020
Positive control + + + 0.100 ± 0.005 0.100 ± 0.005 0.100 ± 0.005 0.100 ± 0.005
2061.66 Da) was homologous to the cysteine membrane
protein Giardia lamblia P15, and another one
(AVPSMKLCIQWSPVRASPCVMLGI, 2587.21 Da)
was homologous to the Planctomycetes bacterium I41
peptides.
The peptides obtained from the culture fluid of
bacteria isolated from natural sources of the Siberian
Federal District were analyzed for antibacterial
properties against Bacillus pumilus and Escherichia
coli. We identified one peptide that exhibited no
antagonistic activity against either gram-negative or
gram-positive bacteria. One peptide fraction showed
high antibacterial properties against both B. pumilus and
E. coli. One peptide was active against E. coli, but not
against B. pumilus (gram-positive bacteria). Finally, four
out of seven peptides under study exhibited moderate
and pronounced antagonism against B. pumilus, but no
antibacterial activity against E. coli.
Our study of the peptides’ antifungal activity
revealed three peptides that could inhibit the growth of
the microscopic fungi Aspergillus niger and Aspergillus
flavus, without stopping it completely (0.1–0.2 mm lysis
zone). Four peptide fractions showed high fungicidal
activity against Aspergillus (0.3–0.5 mm lysis zone).
According to our results, antimicrobial peptides
produced by bacteria isolated from the natural objects of
the Siberian region can be used as promising agents in
the production of pharmaceutical substances and drugs
(after safety trials) to treat infectious diseases, such as
gastrointestinal, respiratory, blood and skin, as well as
fungal infections.
CONTRIBUTION
The authors are equally responsible for the research
results and the manuscript.
CONFLICT OF INTEREST
The authors declare that there is no conflict of
interest.

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