ISSN 1022-7954, Russian Journal of Genetics, 2023, Vol. 59, No. 8, pp. 779–793. © Pleiades Publishing, Inc., 2023.

Russian Text © The Author(s), 2023, published in Genetika, 2023, Vol. 59, No. 8, pp. 898–913.

GENETICS
OF MICROORGANISMS

Reaction of Bifidobacterium longum subsp. infantis Strain

ATCC 15697 to Oxidative Stress
O. V. Averinaa, *, A. S. Kovtuna, D. A. Mavletovaa, R. H. Ziganshinb, and V. N. Danilenkoa
a Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991 Russia
b Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia
*e-mail: olgavr06@mail.ru
Received February 20, 2023; revised March 14, 2023; accepted March 15, 2023

Abstract—The genus Bifidobacterium is one of the most predominant bacterial populations in human-gut
microbiota. Despite the increasing number of studies on the beneficial properties of bifidobacteria for human
health, knowledge about their antioxidant potential is still insufficient. The role of the antioxidant potential
of bifidobacteria in maintaining the homeostasis of the intestinal microbiota of the host organism as a whole
is an important task that requires solutions. For the first time, this paper presents the data of genomic, tran-
scriptome, and proteomic analyses of Bifidobacterium longum subsp. infantis ATCC 15697 strain after the
action of oxidative stress. A growth culture of the strain is exposed to hydrogen peroxide for 2 hours and oxy-
gen for 2 and 4 hours. Preliminary genome analysis of the strain shows the presence of 17 genes encoding a
known protein with antioxidant function, as in other genomes of B. longum subsp. infantis available in the
international database NCBI. Complete transcriptome analysis reveals an increase in the transcript levels by
more than two times for 6 genes with a known antioxidant function. The data of quantitative proteomic anal-
ysis shows an increase in protein levels by more than two times for five enzymes with a known antioxidant
function. Over 28 other proteins with levels increased by more than two times are identified in the cells of the
growth culture in response to the long-term action of oxygen. These proteins can be involved in the processes
of the cell’s response to stress, amino-acid- and nucleotide metabolism, and transport processes. Six proteins
with unknown functions, which may play a significant role in the antioxidant response of anaerobic bifido-
bacteria, are found to have high levels in the cells after the action of stress. The obtained data are supposed to
be used in the selection of B. longum subsp. infantis strains and the creation of pharmabiotics able to correct
the composition of the microbiota.

Keywords: bifidobacteria, genomic analysis, transcriptome analysis, proteomic analysis, oxidative stress
DOI: 10.1134/S1022795423080033

INTRODUCTION deleterious effects including protein misfolding and

The genus Bifidobacterium is one of the predomi-
nant bacterial populations in human gut microbiota.
Their positive functional role for human health is well
characterized [1]. The number of bifidobacteria in
infants born vaginally and breastfed is 90% of the total
intestinal microbiota. Further, the number of bifido-
bacteria in the large intestine of adults decreases to 5%
and decreases even more in the elderly [2]. One of the
important properties of bifidobacteria is resistance to
the action of oxidants and reactive oxygen species
(ROS) and the ability to increase the antioxidant (AO)
and anti-inflammatory status of the host organism
[3–5].
Bifidobacteria are gram-positive microorganisms
from the Actinobacteria class high in G+C. Like most
colon bacteria, bifidobacteria are strict anaerobes, so
oxygen can be an important stressor they need to deal
with. It is known that exposure to oxygen causes the
accumulation of ROS, mainly H2O2, which can cause
aggregation, DNA damage, and lipid peroxidation
leading to cell death. Numerous in vivo and in vitro
studies have demonstrated that bifidobacteria and
their cellular components have an AO ability that pro-
vides a certain degree of protection against oxidative
damage to both their own cells and the cells of their
hosts [5]. Bifidobacteria can exhibit AO activity
through various mechanisms: the chelation of toxic
ions (Fe2+ and Cu 2+ ); the synthesis of AO enzymes,
peptides, and thiols; and compounds with AO proper-
ties [5]. However, AO activity is specific for a strain
and is not common even for a species [6, 7]. Previous
studies have found in the strain Bifidobacterium
longum subsp. longum sensitivity to oxygen (they grow
in the presence of 5% O2 in a liquid culture), and
strains Bifidobacterium longum subsp. infantis and
Bifidobacterium adolescentis hypersensitivity to oxygen
(growth is inhibited under conditions of 5% O2) [8].
However, it has been shown that strains B. longum and

779
780 AVERINA et al.
B. infantis are capable of growth with periodic shaking
on a rocking chair [9]. The genome-sequencing anal-
ysis of B. longum showed the absence of genes for
NADH peroxidase or superoxide dismutase, although
there is a homologue for NADH oxidase [10]; genes
encoding enzymes that reduce oxidative damage are
present, namely alkyl hydroperoxide reductase (ahpC)
[11], thioredoxin reductase [12], and others [5].
In recent years, there has been a growing interest in
bifidobacteria as drugs for the treatment and preven-
tion of various diseases accompanied by the develop-
ment of oxidative stress. This determines the increase
in demand for the use of bifidobacteria as pharmabiot-
ics, dietary supplements, and as part of functional
foods.
The successful application of bifidobacteria
depends not only on scientific studies of their proper-
ties that demonstrate efficacy in providing benefits to
human health, but also on the development of tech-
nologies that ensure survival in large numbers during
industrial cultivation and maintenance during long-
term storage [13]. Damage to ROS cells is one of the
main reasons for the loss of viability of anaerobic pro-
biotics such as bifidobacteria, and therefore has a great
impact on their commercial use.
Despite the growing number of studies on the ben-
eficial functionality of bifidobacteria for human
health [14], knowledge about their AO potential is still
insufficient. In this regard, in the near future, more
and more attention should be paid to studying the
response of bifidobacteria to oxidative stress (OS).
This paper presents for the first time the results of
genomic, transcriptome, and proteomic analyses of
the B. longum subsp. infantis ATCC 15697 strain. The
strain was isolated from the intestine of a child, its
genome was sequenced and well described [15].
B. longum subsp. infantis bacteria are a subspecies of
the Bifidobacterium longum species, i.e., the most
common and stable throughout a person’s life. To
understand the mechanism of the AO defense of cells
B. longum subsp. infantis on OS, we study the growing
culture of the strain under the action of hydrogen per-
oxide for two hours and oxygen for two and four hours.
B. longum subsp. infantis bacteria are dominant in the
gut bifidoflora of infants who are breastfed [16]; there-
fore, deepening knowledge about the AO-defense of
both bifidobacteria cells themselves and their host is of
great importance for the future development of strate-
gies for survival in the composition of infant microbi-
ota. The data obtained are to be used in the selection
of strains of B. longum subsp. infantis and the creation
of pharmabiotics based on them, capable to correct
the composition of microbiota.
RUSSIAN JOURNAL OF GENETICS
MATERIALS AND METHODS
Bacterial Strains, Media, Cultivation Conditions
The culture strain B. longum subsp. infantis ATCC
15697 from the laboratory culture collection was
grown at 37°C in De Man, Rogosa, Sharpe broth
(MRS) supplemented with 0.05% L-cysteine HCl
under anaerobic conditions in a HiAnaerobic System-
Mark III anaerobic jar (HiMedia, India).
Exposure of B. infantis Culture to Oxidative Stress
The culture was grown in liquid MRS medium with
cysteine until the middle of the exponential growth
phase (OD600 0.5–0.6) under anaerobic conditions at
37°C. The control part of the culture was taken before
oxidative stress and after 2 h of incubation under
anaerobic conditions. For the experiment, the culture
after centrifugation (at 28°C and 7500 rpm) and
replacement of the medium with cysteine by the
medium without it were divided into three parts: two
parts were cultivated on a shaker at 250 rpm and 37°C
for two and four hours, and to one part H2O2 was
added to 1 mM and incubated at 37°C under aerobic
conditions for two hours.
mRNA Isolation and Further Sequencing
Cells from the culture fluid (108 cfu/mL) were col-
lected by centrifugation (1 min at 12000 rpm) and the
pellet was resuspended in 100 μL of TE buffer (30-mM
Tris HCl, 1-mM ethylenediaminetetraacetic acid
(EDTA), pH 8.0) supplemented with lysozyme
(20 mg/mL) and incubated for 10 min at 37°C. 350 μL
of lysis buffer (4.5-mM guanidine/HCl, 50-mM Tris
HCl, 30% Triton X-100, pH 6.6) was added to the sus-
pension. The cells were destroyed mechanically with
silica balls (diameter 150–212 mm) on a Speed Mill
plus device (Analytik jena, Germany). RNA was iso-
lated using the RNeasy Mini Kit (Qiagen, United
States) according to the manufacturer’s recommenda-
tions. RNA samples were treated with DNaseI to
destroy DNA residues. The purity of the RNA samples
was tested using a Nano DropTM instrument. Using an
Agilent 2100 bioanalyzer (AgilentRNA 6000 NanoKit),
the total amount of RNA, RIN, 23S/16S was deter-
mined in the samples. The RNA samples were treated
with the Ribo-off rRNA Depletion Kit (Bacteria) to
remove rRNA (16S and 23S) while preserving mRNA.
Next, libraries for sequencing were prepared with the
MGIEasy RNA Directional Library Prep Set accord-
ing to the manufacturer’s protocol. The resulting sin-
gle-stranded circular DNA (ssCirDNA) was synthe-
sized as the final library. The library was amplified
with phi29 polymerase to generate DNA beads. They
were loaded onto a 100 bp pair-ended sequencing
nanochip on the DNBseq (BGI) platform. Sequenced
transcriptome samples were analyzed for read errors
Vol. 59 No. 8 2023
 REACTION OF BIFIDOBACTERIUM LONGUM SUBSP. INFANTIS STRAIN ATCC 15697
using FastQC v 0.11.5 [17]. The sequencing quality was
improved using Trimmomatic v0.39 [18].
Catalog of Orthologs of Genes Encoding
Products with Antioxidant Activity
A list of antioxidants produced by bacteria of the
genus Bifidobacterium was compiled. Then, a search
was made for reference amino-acid sequences for the
genes responsible for the metabolism of the selected
antioxidants. Only sequences that were experimentally
confirmed in research studies were selected. These
reference sequences were then used to search for
orthologs in bacteria of the species B. longum using
BLAST [19] and the NCBI Protein database 2022.
Genome Analysis of B. longum subsp. infantis
To analyze the distribution of genes from the cata-
log of orthologs among bacteria B. longum subsp.
infantis published genomic assemblies were loaded
from the NCBI Assembly database. Only genomes
with the assembly status “complete” were used. The
BlastX program was used to identify homologues of
genes from the reference catalog. The thresholds for
the alignments' filtering were: minimum identity 60%,
minimum relative alignment length 80%.
Analysis of Antioxidant Gene Expression
Analysis of the expression levels of genes responsi-
ble for the antioxidant properties in sequenced sam-
ples of the strain was performed using the collected
catalog of orthologs. Transcriptome reads were
mapped to amino-acid sequences using DIAMOND
v2.0.13 [20]. The hits were filtered using custom scripts
written in the Perl programming language for the fol-
lowing thresholds: ≥60% identity and ≥90% relative
alignment length. Multiple alignments have been fil-
tered out. Thereafter, the number of reads aligned with
each gene was counted and normalized.
Whole Transcriptome Analysis
The reference genomes and their annotations were
downloaded from the RefSeq database by the corre-
sponding ID GCF_001010995.1 for B. longum subsp.
infantis ATCC 15697. Next, hisat2 v2.2.1 [21] was used
for mapping of the quality-checked transcriptome
reads of the sequenced samples. The mappings were
filtered using the SAM tools v1.10 software package
[22]. The read counts were assessed using HTSeq-
count v2.0.2 [23].
Sample Preparation
for Chromato-Mass-Spectrometric Analysis
Cells of the strain B. longum subsp. infantis ATCC
15697 were grown in MRS medium to the exponential
RUSSIAN JOURNAL OF GENETICS Vol. 59 No. 8
781
growth phase (OD600 0.5–0.6). The cells were sepa-
rated from the culture fluid by centrifugation at 7000 rpm
for 30 min, at 4°C. The cell pellet was washed three
times with PBS, pH 7.4. Next, the cells were resus-
pended in a PBS solution, pH 7.4, preincubated at
95°C for 20 min in a ratio of 1 : 10. The cells were then
incubated at 95°C for 10 min. The cells were lysed
using a VibraCell™ Ultrasonic Processor (Sonics,
United States) at a frequency of 20 kHz three times for
15 s with an interval between treatments of 10 s at 4°C.
Fragments of the cell walls were separated by centrifu-
gation at 17000 rpm for 20 min at 4°C. The protein
concentration was determined on a fluorimeter Qubit
2.0 (Invitrogen, United States) as recommended by
the manufacturer.
Hydrolysis of Proteins by Trypsin
Aliquots of cell-lysate solutions containing 20 μg of
protein were dried on a SpeedVac centrifugal vacuum
concentrator (Savant, France) and dissolved in 20 μL
of a buffer solution containing 100-mM Tris pH 8.5,
1% sodium deoxycholate, 10-mM TCEP (Tris(2-car-
boxyethyl)phosphine), and 20-mM 2-CAA (2-chlo-
roacetamide), heated for 20 min at 85°С, cooled to
room temperature, 0.4 μg trypsin in 10-μL 100-mM
Tris pH 8.5 was added, and left to incubate at 37°С
overnight. At the end of incubation, an equal volume
of 2% TFA was added to the reaction mixture, and the
peptides were desalted using an SDB-RPS Stage Tip
microcolumn made from a 200-μL automatic pipette
tip and three pieces of SDB-RPS membrane (3M,
United States) cut with a 16-gauge needle [24]. The
peptide solution was applied to a microcolumn by cen-
trifugation at 300 g, washed with a mixture of solvents
(50 μL of 1% TFA): 50 μL of ethyl acetate (3 times),
50 μL of 0.2% TFA, and eluted with 50 μL of a solu-
tion containing 5% ammonium hydroxide and 80%
acetonitrile in water. The eluate was dried and stored
at ‒80°C. Before analysis, the peptides were dissolved
in 40 μL of a solution containing 0.1% TFA and 2%
acetonitrile in water.
Chromato-Mass-Spectrometry Analysis
Samples were loaded onto a pre-column 50 × 0.1 mm
packed with Inertsil ODS3 3 μm sorbent (GLSciences),
in the solution containing 2% acetonitrile, 98% H2O,
0.1% TFA, at a flow rate of 10 μL/min and separated
at room temperature into a fused silica column 300 ×
0.1 mm with an emitter made on a P2000 Laser Puller
(Sutter, United States) and packed with Reprosil
PURC18AQ 1.9 sorbent (Dr. Maisch). Reversed-
phase chromatography was carried out on an Ultimate
3000 Nano LC System chromatograph coupled to a
QExactive Plus Orbitrap mass spectrometer via a
nanoelectrospray source. For the chromatographic
separation of peptides, a system of solvents A (99.9%
water, 0.1% formic acid) and B (19.9% water, 0.1%
2023
782 AVERINA et al.
formic acid, 80% acetonitrile) was used. Peptides were
eluted from the column with a linear gradient: 3% B 3
min, 3–6% B 2 min, 6–30% B 50 min, 30–55% B 10
min, 55% B 2 min, 55–99% B 0.1 min, 99% B 2 min,
3–99% B 0.1 min at a flow rate of 500 nL/min. Mass-
spectrometry analysis was performed in the DDA
mode (TopN=10) with the following instrument set-
tings: MS1 scan: resolution 70000, scan range 200–
1600 m/z, maximum ion-injection time 35 ms, AGC
level 3 × 106; MS2 scan: 17500 resolution, 30% energy
HCD fragmentation, maximum ion-injection time
80 ms, AGC level 1 × 105.
Analysis of Mass-Spectrometry Data
Mass-spectrometry data were analyzed using the
Peaks studio 10.0 software (Bioinformatics Solutions
Inc.) [22, 25]. Protein identification was performed by
the correlation of tandem mass spectra with a database
of protein sequences. B. longum subsp. infantis ATCC
15697 Uniprot (September 19, 2022) with the follow-
ing parameters: permanent modification of Cys (urea
methylation, variable modifications); deamidation of
Asn/Gln and oxidation of Met; the acceptable level of
false-positive peptide identifications is 0.01 (deter-
mined from the reverse database of amino-acid
sequences); and protease specificity: C-terminus Arg
and Lys (the database search allowed for up to two
missed hydrolysis sites). When identifying peptides,
the deviation of the experimentally obtained mass of
the peptide from its theoretical mass by up to 10 ppm
was allowed, and the deviation of the mass of frag-
ments was up to 0.05 Da.
RESULTS
Genomic Analysis of the Antioxidant Potential
of Bacteria B. longum subsp. infantis
Enzymes and other cellular compounds and
metabolites that respond to oxidative stress in mem-
bers of the B. longum species, were selected after the
analysis of available published data and presented in a
review [5]. Orthologs were selected for genes matched
to proteins with identified antioxidant functionality.
Orthologs were identified as described in the Materials
and Methods section in the genomes of bifidobacteria
species, i.e., human intestinal commensals. The
amino-acid sequences of the selected orthologs were
compiled into a catalog that included 203 sequences
for 27 proteins (Table S1). Amino-acid sequences of
orthologs in FASTA format are available in the data-
base on the website https://github.com/ Alexey-
Kovtun/Catalog. Then, the presence of genes from the
catalog in the complete genomes of various strains of
the B. infantis subspecies was analyzed. Data on the
presence in the genomes of various strains of the sub-
species B. longum subsp. infantis of 17 genes encoding
the subunit C of alkyl hydroperoxide reductase
(ahpC), the subunit F of alkyl hydroperoxide reduc-
RUSSIAN JOURNAL OF GENETICS
tase (ahpF), class-I pyridine nucleotide-disulfide
oxidoreductase, DSBA oxidoreductase, dihydrooro-
tate dehydrogenase, glutaredoxin (grxC2), glutathione
ATP-binding protein (gsiA), linoleic-acid isomerase,
NADH oxidase, P-type ATPase, permease, peroxire-
doxin, thioredoxin domain protein, thioredoxin per-
oxidase, thioredoxin reductase, serine protease are
presented in Table S2. This table includes data on the
genome of the strain B. longum subsp. infantis ATCC
15697, which also revealed the presence of these genes,
except for the gene encoding serine protease.
To determine the functionality of the identified
genes and other genes with antioxidant properties, a
general transcriptome analysis of the growing culture
of the strain B. longum subsp. infantis ATCC 15697,
exposed to H2O2 and oxygen, was carried out.
Transcriptome Analysis of the Antioxidant Response
of the Strain B. longum subsp. infantis ATCC 15697
For complete transcriptome analysis, a culture of
the strain B. longum subsp. infantis ATCC 15697 in the
exponential-growth phase, which was exposed to
hydrogen peroxide for two hours and oxygen for two
and four hours, was used. These conditions were used
to get closer to the environment of bifidobacteria in
the intestinal tract with possible oxidative stress during
inflammatory processes or during industrial processes
for the production of a bacterial preparation.
Total RNA was isolated from culture cells taken
before (control) and after exposure to oxidative stress
and sequenced on the DNBSEQ System after sample
preparation. Transcriptome samples of the strain
B. longum subsp. infantis ATCC 15697 were sequenced
as paired 100 bp end reads. The complete parameters
of the samples are presented in Table S3. The quality-
control procedure showed that the samples were suit-
able for analysis and their dimensions did not decrease
significantly after trimming. The total amount of data
is almost evenly distributed across the samples. Tran-
scriptome reads were aligned to the reference catalog
using DIAMOND and filtered as described in the
Materials and Methods section. The number of
remaining reads used to estimate expression levels was
466051, 439089, 316276, 523810 and 375600. Tran-
scriptome reads have been mapped on the genome
B. longum subsp. infantis ATCC 15697 using HTSeq-2.
The total number of reads used to further evaluate
expression levels, amounted to 28334402 (control 1),
28733996 (control 2), 27525358 (H2O2, 2 h),
28459072 (O2, 2 h) and 26529750 (O2, 4 h).
The total number of genes with identified tran-
scripts was 2647. Of these, a more than 2-fold increase
in levels was found for transcripts of 243 genes in
response to H2O2, 67 genes in response to the action of
O2 within two hours and 99 genes in response to the
Vol. 59 No. 8 2023
 REACTION OF BIFIDOBACTERIUM LONGUM SUBSP. INFANTIS STRAIN ATCC 15697
Table 1. Changes in the levels of transcripts of genes encoding antioxidant-response products in B. infantis ATCC 15697,
before and after oxidative stress
Gene product
Protein with thioredoxin domain
Thioredoxin reductase
P-type ATPase
Linoleic-acid isomerase
Class-1 pyridine nucleotide-disulfide oxidoreductase
Fe-permease
* Fold ratio to the abundance across different samples before and after oxidative stress.
action of O2 within four hours. Using the catalog of
orthologs to search for genes encoding products with
antioxidant activity, six different genes were identified
among the transcripts, which are presented in Table 1.
In general, oxidative stress affected a more than 2-fold
increase in the transcript levels for genes encoding thi-
oredoxin domain protein, thioredoxin reductase,
P-type ATPase, linoleic-acid isomerase, and class-1
pyridine nucleotide-disulfide oxidoreductase. The
transcripts of genes encoding thioredoxin reductase
were increased by 9.27 times after exposure to H2O2
and by 2.13 times after the action of O2 within four
hours of incubation. The transcripts for the protein
with the thioredoxin domain are increased by 2.66
times after H2O2 exposure. All stress factors provoked
an increase in the P-type ATPase transcript by 19.92
times upon H2O2 exposure, 2.05 times upon the action
of O2 for two hours, and 3.68 times upon the action of
O2 for four hours of incubation. The transcripts for the
gene encoding linoleic-acid isomerase increased by
5.31 times upon exposure to only H2O2. The tran-
scripts for the gene encoding class-1 pyridine nucleo-
tide-disulfide oxidoreductase increased by 2.4 and
1.71 times and for the permease gene increased by 2.57
and 2.54 times only when exposed to O2.
To identify the translation products of the studied
genes under conditions of oxidative stress, quantitative
proteomic analysis of the cell culture of the strain
B. infantis ATCC 15697 was employed.
Quantitative Proteomic Analysis of the Antioxidant
Response of the B. infantis ATCC 15697 Culture
For proteomic analysis, we also used a growing cul-
ture of the strain B. infantis ATCC 15697 in the expo-
nential-growth phase, which was exposed to hydrogen
peroxide for two hours and oxygen for two and four
hours.
Proteins were isolated from culture cells selected
before (control) and after exposure to oxidative stress
RUSSIAN JOURNAL OF GENETICS Vol. 59
783
Locus_tag *H2O2 *O2 *O2
gene 2 hours/control 2 hours/control 4 hours/control
BLON_RS01630 2.66 1.06 1.79
BLON_RS12925 9.27 1.41 2.13
BLON_RS01320 19.92 2.05 3.68
BLON_RS04150 5.31 1.47 0.79
BLON_RS08540 0.83 2.4 1.71
BLON_RS01040 0.45 2.57 2.54
and studied using quantitative proteomic analysis
described in the Materials and Methods section. Vol-
cano plot for proteins of B. infantis ATCC 15697 in
Fig. 1 shows the number of proteins with a detected
difference (increase or decrease) after the action of
oxidative stress. Under the influence of H2O2 a differ-
ence is found for two proteins; under the action of O2
for two hours, a difference is found for six proteins;
and under the action of O2 for four hours, a difference
is found for 63 proteins. Further analysis included only
data on an increase in the amount of protein by more
than 2 times.
Five known proteins with AO functionality have
been identified in response to stress (Table 2). An
increased level glutaredoxin was observed (by 3.55
times) only when exposed to oxygen during four hours
of incubation. However, the level of transcripts of the
gene encoding it was at a low level. P-type ATPase and
ferritin increased after the action of all stress factors, as
well as their transcripts (Tables 1 and 2). The quantity
of Fe-permease and subunits of Piridoxal-5'-phos-
phate synthase PdxT increases only under the action of
oxygen by 30.5 times (2 h), 24.7 times (4 h), and 3.3
times (4 h), respectively.
The remaining 34 identified proteins with an
increased level in cells after the action of oxidative
stress perform various functions, but mainly protective
ones.
Under the action of H2O2 an increase was found
only for the helix-turn-helix transcription regulator by
5.26 times, as well as the transcript by 9.6 times; of
extracellular solute-binding proteins from family 1 by
4.35 fold with an increase in the transcript level by
21.44 fold, and from family 5 by 9.09 fold without a
revealed increase in the transcripts and one protein
with unknown functions with a 25-fold increase and a
30.15-fold increase in the transcript. The action of O2
for two hours provoked an increase in only one protein
with unknown functions by 14.58 times and an
No. 8 2023
784 AVERINA et al.

(a) (b)

42.5 27.5
40.0 25.0
37.5
35.0 22.5
32.5 20.0
30.0
Significance

Significance
27.5 17.5
25.0 15.0
22.5
20.0 12.5
17.5
15.0 10.0
12.5 7.5
10.0
7.5 5.0
5.0 2.5
2.5

1/ 1/ 1/ 1/ 1/ 1 2 4 8 16 32 1/ 1/ 1/ 1/ 1 2 4 8 16 32 64
32 16 8 4 2 16 8 4 2
Ratio Ratio
(c)
200
190
180
170
160
150
140
130
Significance

120
110
100
90
80
70
60
50
40
30
20
10

1/ 1/ 1/ 1/ 1 2 4 8 16 32
16 8 4 2
Ratio

Fig. 1. The volcano plot for proteins of B. infantis ATCC 15697, induced at a stress response by the action of: (a) H2O2 (b) O2
during 2 h of incubation, (c) O2 during 4 h of incubation.

increase in the transcript by 2.3 times. The remaining
proteins increased in level more than 2-fold after pro-
longed exposure to oxygen. However, for most of
them, a low level of transcription was observed.
DISCUSSION
From the few published data for bifidobacteria,
enzymes, and other compounds with AO action and
genes encoding them were selected [5]. A search was
made for the orthologs identified for them in the
genomes of strains of the subspecies B. longum subsp.
infantis. The study of the distribution of AO genes
showed the presence of 17 in most genomes, which
indicates their conservatism and, possibly, common
mechanisms of AO-defense in B. longum subsp. infan-
tis. However, in bifidobacteria, strain-specificity in
the manifestation of AO ability was revealed [5]. It is
possible that other unknown enzymes and metabolites
are also involved in protection against oxidative stress
in bifidobacteria. To determine the functionality of

RUSSIAN JOURNAL OF GENETICS Vol. 59 No. 8 2023

 REACTION OF BIFIDOBACTERIUM LONGUM SUBSP. INFANTIS STRAIN ATCC 15697 785

Table 2. Comparative data of proteomic and transcriptome analysis of cells of the strain B. infantis ATCC 15697 before and
after exposure to oxidative stress
Locus_tag H2O2 O2 O2
Gene product
gene 2 hours/control 2 hours/control 4 hours/control
Antioxidant response
Glutaredoxin BLON_RS12860 NS NS 3.55*
0.49 1.55 1.05**
Ferritin BLON_RS00160 4.54 NS 5.88
21.61 1.70 3.57
Fe-permease BLON_RS01040 NS 30.50 24.70
0.45 2.57 2.54
P-type ATPase BLON_RS01320 3.57 NS 2.10
19.92 2.05 3.68
Pyridoxal-5'-phosphate synthase BLON_RS10350 NS NS 3.30
subunit pdxT
0.81 2.36 3.17
Response to stress
Extracellular ligand-binding receptor BLON_RS02930 NS NS 3.49
0.35 1.0 1.58
Extracellular ligand-binding receptor BLON_RS03810 NS NS 2.18
1.33 2.83 2.0
RelB antitoxin BLON_RS01740 NS NS 2.98
2.5 0.82 1.0
Ribosome maturation factor rimM BLON_RS01970 NS NS 2.68
0.66 1.0 1.27
Transcription elongation factor greA BLON_RS09225 NS NS 2.62
1.68 1.95 2.3
FMN Domain-Containing Protein BLON_RS01070 NS NS 2.39
1.56 1.0 0.36
DnaJ domain of heat shock protein BLON_RS00750 NS NS 2.29
10.3 0.8 4.2
Tyrosine-tRNA-ligase BLON_RS09710 NS NS 2.27
0.91 0.57 0.54
ClpB chaperone protein BLON_RS12265 NS NS 2.18
21.57 1.58 8.1
Endoribonuclease L-PSP BLON_RS01645 NS NS 2.64
0.59 1.75 2.5
Amino-acid metabolism
Alpha subunit tryptophan synthase BLON_RS07130 NS NS 2.19
0.63 2.1 2.0

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786 AVERINA et al.

Table 2. (Contd.)
Locus_tag H2O2 O2 O2
Gene product
gene 2 hours/control 2 hours/control 4 hours/control
Anthranilate phosphoribosyltransferase BLON_RS04830 NS NS 2.13
0.79 2.03 3.41
3-Isopropylmalate dehydrogenase BLON_RS10735 NS NS 2.04
0.57 1.92 2.63
Threonine aldolase BLON_RS10060 NS NS 2.02
0.42 0.46 0.38
Metabolism of nucleotides
Nucleoside 2-deoxyribosyltransferase BLON_RS03085 NS NS 2.22
0.47 1.1 1.65
Phosphoribosylamine glycine ligase BLON_RS10180 NS NS 2.77
1.5 0.85 0.33
Transcription regulation
Helix-turn-helix transcription regulator BLON_RS04795 5.26 NS 6.95
9.6 1.84 1.0
Transportation
Extracellular solute-binding protein BLON_RS12635 4.35 NS 2.80
(family 1)
21.44 0.44 0.44
Extracellular solute-binding protein BLON_RS10690 NS NS 3.33
(family 1)
0.58 1.16 1.16
Extracellular solute-binding protein BLON_RS01440 9.09 NS NS
(family 5)
0.9 1.25 0.92
Extracellular solute-binding protein BLON_RS10445 NS NS 6.91
(family 5)
1.55 2 0.86
Extracellular solute-binding protein BLON_RS04415 NS NS 2.10
(family 5)
0.5 1.96 2.0
Extracellular solute-binding protein BLON_RS03845 NS NS 2.96
(family 3)
1.12 0.82 0.82
UDF-N-acetylglucosamine-N-acetyl- BLON_RS04355 NS NS 6.03
muramyl (pentapeptide)-pyrophos-
0.93 1.14 0.83
phoryl-undecaprenol-N-acetylglucos-
amine transferase
SecE subunit of the translocase BLON_RS06530 NS NS 2.48
0.6 0.6 0.46

RUSSIAN JOURNAL OF GENETICS Vol. 59 No. 8 2023

 REACTION OF BIFIDOBACTERIUM LONGUM SUBSP. INFANTIS STRAIN ATCC 15697 787

Table 2. (Contd.)
Locus_tag H2O2 O2 O2
Gene product
gene 2 hours/control 2 hours/control 4 hours/control
ABC-2-transporter BLON_RS12655 NS NS 2.36
6.17 1.57 1.02
ABC-transporter BLON_RS01065 NS NS 3.84
1.19 1.25 0.47
Fsx Domain-Containing Protein BLON_RS01060 NS NS 2.47
0.9 1.25 0.63
Unknown proteins
Protein with unknown functions BLON_RS01045 NS 14.58 14.56
0.65 2.3 1.38
Protein with unknown functions BLON_RS10130 25.0 NS NS
30.15 1.5 0.75
Protein with unknown functions BLON_RS11705 NS NS 13.33
0.65 0.54 0.27
Protein with unknown functions BLON_RS01055 NS NS 5.49
0.77 1.55 0.96
Protein with unknown functions BLON_RS12535 NS NS 2.39
0.8 1.66 3.14
Protein with unknown functions BLON_RS07940 NS NS 2.33
2.1 1.46 2.5
* Fold ratio to the average abundance of proteins across different samples before and after oxidative stress; ** fold ratio to the abundance
of transcripts across different samples before and after oxidative stress. NS means no change in levels.

the AO genes identified in the genomes and identify
new genes involved in the response to oxidative stress,
we applied an integrated approach using omics tech-
nologies. Analysis of the total transcriptome showed
an increase in transcript levels for six genes encoding
known AO proteins. After the action of all oxidative
factors in B. infantis ATCC 15697, a high level of tran-
scripts of the gene encoding P-type ATPase was deter-
mined. It was reported that the gene zntA1, encoding
P-type ATPase, increased by 2.01 times after 60 min of
oxygen exposure of the strain B. longum BBMN68
[26]. P-type ATPases may be involved in Mn2+ trans-
fer, which then removes superoxide anions in bifido-
bacteria cells [12]. Mn2+ not only replaces superoxide
dismutase in the removal of superoxide anions, but
can also remove H2O2 [27]. After exposure to H2O2
and O2 the level thioredoxin reductase and a protein
with a thioredoxin domain. The genes encoding glu-
taredoxin, thioredoxin, and thioredoxin reductase
increased by more than 6 times in 60 min when the
strain B. longum BBMN68 was exposed to oxygen
[26]. The thioredoxin-dependent repair system plays
an important role in the response to oxidative stress by
directly reducing H2O2, the removal of hydroxyl radi-
cals, the suppression of singlet oxygen, and the main-
tenance of intracellular thiol-disulfide balance [28].
The action of oxygen had an effect on an increase in
the level of Fe-permease. Fe-permeases in the plasma
membrane are involved in the transfer of iron [29].
A significant increase in the level of the transcript of
the gene encoding linoleic-acid isomerase was noted
only after exposure to hydrogen peroxide. Conjugated
linoleic acid, produced by some species of bifidobac-
teria, does not itself possess AO properties, but its
metabolites are able to protect cells from harmful oxi-
dative effects [30, 31]. After exposure to oxygen there
an elevated level of class-1 pyridine nucleotide-disul-
fide oxidoreductase gene transcript was noted in the cells

RUSSIAN JOURNAL OF GENETICS Vol. 59 No. 8 2023

788 AVERINA et al.
of B. infantis ATCC 15697. This enzyme is known to par-
ticipate in the response of cells to oxidative stress [11].
Quantitative proteomic analysis revealed other
proteins with AO functions. After the action of H2O2
the amount of ferritin increased by 4.54 times. Ferritin
catalyzes the oxidation of Fe2+ ions with hydrogen
peroxide, which prevents the formation of a hydroxyl
radical by the Fenton reaction [12]. At the level of
translation, as well as at the level of transcription,
P-type ATPase increases under the action of all stress
factors, which indicates the important role of the pro-
tein in protecting against oxidative stress in B. infantis.
It is important to note a significant increase in the AO
of Fe-permease, pyridoxal-5'-phosphate synthase
(PdxT subunits) and glutaredoxin with prolonged
exposure to oxygen. Pyridoxal-5'-phosphate synthase
participates in the synthesis of vitamin B6. Vitamin B6
plays an important role in the antioxidant mechanism
and is a glutathione cofactor [32]. Glutaredoxin acts as
an antioxidant, reducing dehydrocarbonate, peroxire-
doxins, and methionine sulfoxide reductase [33]. The
absence of elevated levels of AO proteins detected by
transcriptome analysis can be explained by their instabil-
ity. It is possible that they are rapidly destroyed, and it is
difficult to fix them by the method of analysis used [34].
The other identified proteins with elevated levels in
the cells are involved in stress response processes,
amino acid and nucleotide metabolism, and transport
processes. For the strain B. infantis ATCC 15697,
stress proteins ClpB chaperone and heat-shock pro-
tein with the DnaJ domain increase mainly after pro-
longed exposure to oxygen. The transcripts of genes
encoding the DnaJ and ClpB chaperones increased in
the strain B. longum BBMN68 after 60 min of oxygen
exposure [26]. ClpB, together with DnaK, DnaJ, and
GrpE, is involved in the suppression of protein aggre-
gation. This is a universal phenomenon found in the
responses of various organisms to various abiotic-
stress conditions [35]. The participation of ClpB in
antioxidant activity is assumed in the strain Limosilac-
tobacillus fermentum U-21, which has a high antioxi-
dant potential in the model of parkinsonism [36]. In
the cells of the strain B. infantis ATCC 15697 an excess
of RelB antitoxin, the protein with the FMN domain,
tyrosine-tRNA ligase, and endoribonuclease L-PSP
was also found. RelB antitoxin neutralizes the action
of the toxin formed during cell stress [37]. Tyrosine
tRNA ligase forms carbon–oxygen bonds. Endoribo-
nuclease L-PSP breaks down RNA in the cell. The
FMN domain was found in common stress proteins
[38]. It can help bacteria respond to oxidative stress.
Thus, these proteins may play an important role in
protecting bifidobacteria from oxidative stress.
RUSSIAN JOURNAL OF GENETICS
Under prolonged exposure to oxygen, the levels of
proteins involved in the metabolism of nucleotides and
in the metabolism and transport of amino acids
increased. Genes belonging to the category COG E
(transport and metabolism of amino acids) were
induced compared to the control in the strain B. longum
BBMN68 upon exposure to oxygen [26], which indi-
cates that the processes of amino-acid- and protein
biosynthesis, transport, and metabolism are enhanced
under oxidative stress. In the strain B. infantis ATCC
15697 the level of 11 different proteins with transport
functions increases mainly during long-term cultiva-
tion on a shaker. The activity of the transport system is
switched on in response to the needs of cells both in
compounds with antioxidant properties and in sources
of additional energy necessary to restore intracellular
balance.
In the cells of B. infantis ATCC 15697 after pro-
longed exposure to oxygen, the level of six proteins
with unknown functions increased, indicating that the
cells use unknown mechanisms of protection against
oxidative stress. These proteins are of great interest for
further research.
Thus, it can be concluded that the mechanism of
antioxidant protection in the studied strain includes
both known mechanisms characteristic of bacteria of
the species B. longum, namely, the synthesis of AO
enzymes, compounds with AO properties, the chela-
tion of toxic ions (Fe2+ ), as well as specific mecha-
nisms, including both intracellular mechanisms of
general anti-stress protection and unknown pathways
that may involve proteins with unknown functions.
The study of the role of selected strains of bifido-
bacteria in ensuring the formation of the anti-inflam-
matory potential of the microbiota and the host organ-
ism is undoubtedly a promising area of research [4].
FUNDING
This work was supported by the Russian Foundation for
Basic Research, project no. 20-54-18006. The participation
of V.N. Danilenko and O.V. Averina was also funded under
government task no. 0092-2022-0003.
COMPLIANCE WITH ETHICAL STANDARDS
The authors declare that they have no conflicts of interest.
This article does not contain any studies involving ani-
mals or human participants performed by any of the
authors.
Vol. 59 No. 8 2023
 REACTION OF BIFIDOBACTERIUM LONGUM SUBSP. INFANTIS STRAIN ATCC 15697 789

APPENDIX
Table S1. Reference catalog of amino-acid sequences encoded by genes with antioxidant properties in bacteria of the Bifido-
bacterium genus
Enzyme name Number of orthologs

Alkyl hydroperoxide reductase ahpC 6

Alkyl hydroperoxide reductase ahpF 6

Class-I pyridine nucleotide-disulfide oxidoreductase 3

Cobalamin-independent methionine synthase II 9

DSBA oxidoreductase 9

Dihydroorotate dehydrogenase 9

Glutaredoxin grxC2 (nrdH) 7

Glutathione import ATP-binding protein gsiA 8

Hydroxycinnamic acid esterase caeA—Carboxylesterase A 4

Linoleic-acid isomerase 5

NADH oxidase 11

Oxygen-dependent coproporhpyrinogen III oxidase 9

P-type ATPase 9

Peptidase O pepO 9

Permease 5

Peroxiredoxin 5

Polyphosphate kinase ppk 9

Pyrophosphohydrolase mutT1 9

Ribonucleotide reductase nrdA 6

Subtilisin-like serine protease 2

Superoxide dismutase sodB 12

Superoxide dismutase sodC 11

Thioredoxin 4

Thioredoxin domain protein 9

Thioredoxin peroxidase 7

Thioredoxin reductase 14

Thioredoxin reductase-like protein 6

RUSSIAN JOURNAL OF GENETICS Vol. 59 No. 8 2023

 Table S2. Distribution of genes encoding products with antioxidant functionality in the genomes of Bifidobacterium longum subsp. infantis
790

Strain

Alkyl hydroperoxide
reductase ahpC
Alkyl hydroperoxide
reductase ahpF
Class-I pyridine
nucleotide-disulfide
oxidoreductase
Oxidoreductase DSBA
Dihydroorotate
dehydrogenase
Glutaredoxin grxC2
Glutathione
ATP-binding protein gsiA
Linoleic-acid
isomerase
NADH-oxidase
P-type ATPase

B. longum subsp. infantis 157F + + + + + + + + + +

B. longum subsp. infantis ATCC 15697 = + + + + + + + + + +

JCM 1222 = DSM 20088

B. longum subsp. infantis isolate USA001 1 + + + + + + + + + +

B. longum subsp. infantis strain BINF + + + + + + + + + +

B. longum subsp. infantis strain BT1 + + + + + + + + + +

AVERINA et al.

B. longum subsp. infantis strain CECT 7210 + + + + + + + + + +

B. longum subsp. infantis strain JCM 11347 + + + + + + + + + +

B. longum subsp. infantis strain JCM 11660 + + + + + + + + + +

RUSSIAN JOURNAL OF GENETICS

B. longum subsp. infantis strain JCM 7009 + + + + + + + + + +

Vol. 59
B. longum subsp. infantis strain JCM 7010 + + + + + + + + + +

No. 8
B. longum subsp. infantis strain KCTC 5934 + + + + + + + + + +

B. longum subsp. infantis strain NCTC11817 + + + + + + + + + +

2023
 Table S2. (Contd.)

Strain

Permease
Peroxiredoxin
Serine protease
Protein
with the thioredoxin
domain
Thioredoxin
peroxidase
Thioredoxin
reductase
Thioredoxin-
reductase-like
protein

B. longum subsp. infantis 157F – + + + + + +

B. longum subsp. infantis ATCC 15697 = + + – + + + +

RUSSIAN JOURNAL OF GENETICS

JCM 1222 = DSM 20088

Vol. 59
B. longum subsp. infantis isolate USA001 1 + + – + + + +

B. longum subsp. infantis strain BINF + + – + + + +

No. 8
B. longum subsp. infantis strain BT1 + + – + + + +

2023
B. longum subsp. infantis strain CECT 7210 – + + + + + +

B. longum subsp. infantis strain JCM 11347 + + – + + + +

B. longum subsp. infantis strain JCM 11660 – + + + + + +

B. longum subsp. infantis strain JCM 7009 + + – + + + +

B. longum subsp. infantis strain JCM 7010 + + – + + + +

B. longum subsp. infantis strain KCTC 5934 – + + + + + +

REACTION OF BIFIDOBACTERIUM LONGUM SUBSP. INFANTIS STRAIN ATCC 15697

B. longum subsp. infantis strain NCTC 11817 + + – + + + +

791
792 AVERINA et al.
Table S3. Parameters of analyzed transcriptome samples
Before trimming
Sample name
size, million reads size, gigabase
BIN1 30.4
BIN2 30.6
BIN3 30.5
BIN4 30.5
BIN5 28.2
BIN1 is control 1; BIN2 is control 2; BIN3 is H2O2, 2 h; BIN4 is O2, 2 h; BIN5 is O2, 4 h.
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