The complete genome sequence of Bifidobacterium animalis subsp. lactis 01 and its integral components of antioxidant defense system

Abstract

The strain Bifidobacterium animalis 01, isolated from centenarians, showed promising antioxidant potential in our previous studies. In this study, the genome information on strain 01 and the important antioxidant components are presented. The complete genome comprises a single circular chromosome (1,931,632 bp; 60.49% G + C content) with 1569 coding DNA sequences, 52 tRNA, and 9 rRNA operons. Based on phylogenomic analyses, strain 01 was designated as B. animalis subsp. lactis 01. The genomic analysis reveals that at least eight protein-coding genes are antioxidant-related genes. The conditions for simulating the oxidative stress have been determined. The results of quantitative reverse transcription PCR further demonstrated that the genes encoding the thioredoxin system (ahpC, ahpF, bcp, trxB, trxA, nrdH, and msrAB) and non-enzyme factors of the divalent cation transporter gene (mntH) were upregulated under the H₂O₂ challenge, indicating that the eight genes were effective components of the antioxidant system. The results of this study could benefit for understanding the antioxidant mechanism of B. animalis 01 and future utilization of it as a potential antioxidant agent.

Electronic supplementary material

The online version of this article (10.1007/s13205-019-1890-6) contains supplementary material, which is available to authorized users.

Introduction

Reactive oxygen species (ROS) are produced by aerobic respiration and immune defense of organisms (Lü et al. 2010). The excessive amount of ROS can result in cellular damage, which promotes chronic diseases, such as cardiovascular diseases, diabetes, and cancer (Stephens et al. 2009; Sosa et al. 2013). The consumption of antioxidant supplements has been proposed to alleviate ROS and presumed beneficial for human health (Lobo et al. 2010). It has been shown that probiotic strains present significant antioxidant abilities (Mishra et al. 2015; Amaretti et al. 2013). In addition, certain probiotics can act as antioxidants to maintain intestinal redox balance in the gut by adhering to the intestinal lumen and colonizing the intestine (Tang et al. 2018).

A number of recent studies have reported that Bifidobacterium spp. showed great antioxidative activities (Mishra et al. 2015). The antioxidant capacity of B. animalis subsp. lactis INL1 was evaluated by 2,2ʹ-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) and ·OH technique. The different fractions, such as culture supernatant and lysate supernatant, exhibited high radical scavenging activity with both radicals (Loyeau et al. 2018). In B. longum, silent information regulator 2 could positively regulate the activity of its antioxidant enzymes (Guo et al. 2017). B. bifidum ATCC 29521 could decrease the intracellular level of ROS, and its incubated extracts also showed antioxidative activity by chelating metal ions (Wang et al. 2016). B. subsp. lactis DSMZ 23032 showed an antioxidative capacity with total antioxidant activity, trolox equivalent antioxidant capacity, and total glutathione values (Amaretti et al. 2013). Therefore, these species are potential candidates of natural antioxidant bioresource to promote the human health.

B. animalis 01 was isolated from feces of healthy centenarian volunteers in Guangxi, China. B. animalis 01 itself and its protein extracted have been observed to possess antioxidant activity both in vitro and in vivo (Zhang et al. 2009; Shen et al. 2010, 2011). However, the antioxidant mechanism of this strain is still largely unknown. In this study, to further investigate its antioxidant mechanism, the complete genome information of strain 01 and its integral components of antioxidant defense system are presented. The results of this study lay the theoretical foundation for the future application of strain 01 in the prevention of oxidative stress-related disorders.

Methods

Genome sequencing, assembly, and annotation

The genomic DNA of strain 01 was extracted using the QIAGEN DNA Extraction Kit according to the manufacturer’s instruction (Qiagen, CA, USA). Genome sequencing was performed by the Illumina Hiseq 2000 platform (2 × 100 bp). After sequencing, the short reads were assembled by SOAPdenovo v2.04 (http://soap.genomics.org.cn) (Luo et al. 2012).

After gap closing by SOAP GapCloser, a draft genome with 23 scaffolds was achieved. Gaps between scaffolds were closed by polymerase chain reaction (PCR) and Sanger sequencing. Genome annotation was applied by RAST (Overbeek et al. 2008). COG and Pfam (http://pfam.xfam.org/) were used to predict functional genes. The genes related to antioxidant activities were identified from the genome of B. animalis 01 using BLAST from NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

H₂O₂ treatment of B. animalis subsp. lactis 01

As the most stable ROS, H₂O₂, which can diffuse through cells and form other active ROS, is generated from nearly all sources of oxidative cycle. Thus, H₂O₂ was added to the cultured strain 01 to induce the oxidative stress. Strain 01 was anaerobically cultured in De Man, Rogosa, and Sharpe (MRS) broth (AoBoxing, Beijing, China) at 37 °C until an OD600 of ~ 1.1, after which H₂O₂ was supplemented in a series (0.5, 1.0, 1.5, 2.0, and 2.5 mM), with the control containing no H₂O₂. The samples were collected at 0, 30, and 60 min after H₂O₂ addition and immediately diluted and plated on MRS with 0.05% cysteine agar. The plates were incubated anaerobically at 37 °C for 48 h before enumeration. The replicates were prepared in duplicate.

Determination of intracellular ROS

The production of intracellular ROS was measured using flow cytometry with 2′,7′-dichloro-uorescein diacetate (DCFH-DA) staining, as described previously (Li et al. 2017). After treatments with 1.5 mM H₂O₂ for 0, 30, and 60 min, cells were washed twice with phosphate buffer saline (PBS) (pH 7.2), and then, DCFH-DA (10 μM) was added to the cells for 30 min at 37 °C. The cells were washed with PBS to remove extracellular DCFH-DA and resuspended in PBS. The intracellular ROS levels were measured by FACSCalibur flow cytometry (BD Biosciences, USA) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Real-time quantitative PCR (RT-qPCR) analysis

B. animalis subsp. lactis 01 was cultured as described above, after which H₂O₂ was supplemented at the selected concentration for 0 (T0), 5 (T1), 30 (T2), or 60 min (T3). The total RNAs of different samples were isolated using the Trizol reagent (Invitrogen, United States) following the manufacturer’s instruction. To completely eliminate the DNA, the RNA samples were treated with 50 µg/mL RNase-free DNaseI (Takara, Japan). The quality of isolated RNAs was evaluated by gel electrophoresis. The RNA samples were reverse transcribed into single-stranded cDNA using PrimeScript first-strand cDNA Synthesis Kit (Takara, Japan). RT-qPCR was performed using 7500 Fast Real-Time PCR system (Applied Biosystems) with SYBR FAST qPCR Kit (Kapa Biosystems, USA). The primers sequences for RT-qPCR are listed in Table S1. The qPCR data were analyzed by the 2^−ΔΔCT method (Livak and Schmittgen 2001). And the 16S rRNA gene was used as an internal reference. Table S1 lists the gene-specific primer sequences.

Availability of data and materials

The complete genome sequence of B. animalis subsp. lactis 01 was deposited at GenBank under the accession number CP035497. This strain has been deposited in China Center of Industrial Culture Collection under the accession number CICC no. 24193. The community metadata standards the “Minimal Information about any (X) Sequence” (MixS), which is shown in Table 1.

Table 1.

The community metadata standards the “Minimal Information about any (X) Sequence” (MixS) of Bifidobacterium animalis subsp. lactis 01

Investigation
Investigation type	investigation_type		BA
Project name	project_name		PRJNA516982 Bifidobacterium animalis strain:01
Environment
Collection date	collection_date		Missing (before 2000)
Geographic location (latitude and longitude)	lat_lon
Geographic location (country and/or sea, region)	geo_loc_name		China: Guangxi
Environment (biome)	biome		Homo sapiens
Environment (feature)	feature		Gut
Environment (material)	material		Feces
MIMS/MIENS extension
Environmental package	env_package		Feces
Depth	depth	Meter	1
Elevation	elev	Meter	0
Nucleic acid sequence source
Number of replicons	num_replicons		3
Reference for biomaterial	ref_biomaterial		10.1016/j.anaerobe.2010.06.006; 10.1007/s00284-010-9827-7; 10.1016/j.foodchem.2008.12.006
Observed biotic relationship	biotic_relationship		Free-living and particle-associated
Trophic level	trophic_level		Heterotroph
Relationship to oxygen	rel_to_oxygen		Anaerobes
Isolation and growth condition	isol_growth_condt		B. animalis subsp. lactis 01 grows optimally at a range of 37 °C, a pH of 6.5; culture media: MRS
Sequencing
Sequencing method	sequencing_meth		Illumina
Assembly	assembly		Assembler: SOAPdenovo v2.04
Finishing strategy	finishing_strategy		Statuts: finished;
Relevant electronic resources	url		http://www.ncbi.nlm.nih.gov/genomeprj/CP035497

Results and discussion

General features

The complete genome of B. animalis 01 contains a circular chromosome of 1,931,632 bp and no plasmid, with G + C contents of 60.49%. The chromosome contains 1569 protein-coding genes, 52 tRNA, and 9 rRNA genes (Fig. 1). The identified genes were classified into 21 functional categories according to Clusters of Orthologous Groups (COG) of protein designation (Tatusov et al. 2003) (Table S2). Strain 01 was known as B. animalis 01 in our previous studies (Zhang et al. 2009; Shen et al. 2010, 2011). However, on the basis of the comparative phylogenomic analysis of B. animalis genomes, strain 01 should be now reassigned to denote B. animalis subsp. lactis 01 (Fig. S1).

Fig. 1.

Circular genome map of Bifidobacterium animalis subsp. lactis 01. Circles are shown from the outside to inner. Ring 1, Genome sequences. Ring 2 and 3, COG annotated coding sequences. Ring 4, KEGG enzymes. Ring 5, RNA genes. Ring 6, GC content. Ring 7, GC skew. Very short features were enlarged to enhance visibility. Clustered genes, such as several rRNA genes, may appear as one line due to space limitations. The image was created by software Circos

Identification of gene-coding antioxidant system

According to the annotation of genome, eight antioxidant-related genes of strain 01 were identified (Table 2), included alkyl hydroperoxide reductase subunits C and F (ahpC and ahpF), bacterioferritin comigratory proteins (bcp), thioredoxin reductase (trxB), thioredoxin (trxA), glutaredoxin-like proteins (nrdH), peptide methionine sulfoxide reductase (msrAB), and divalent metal cation transporter (mntH). Among these genes, ahpC, bcp, ahpF, trxB, trxA, nrdH, and msrAB are the basic components of the thioredoxin (Trx) system, which is a functional antioxidant system in protecting cells from oxidative damage (Lu and Holmgren 2014). MntH actively acquires a high-affinity manganese. The involvement of manganese is critical for defensing against ROS (Huang et al. 2017).

Table 2.

Antioxidant-related genes in the B. animalis subsp. lactis 01 genome

COG category	Proposed function	Gene name	Locus tag(s)	Coordinatesc	Protein size
Oa	Alkyl hydroperoxide reductase subunit C	ahpC	ET527_04440	994497..995060(+)	187aa
O	Alkyl hydroperoxide reductase protein F	ahpF	ET527_04445	995079..996851(+)	590aa
O	bacterioferritin comigratory proteins	bcp	ET527_04555	1021161..1021646(+)	161aa
O	Thioredoxin reductase	trxB	ET527_08195	1923658..1924620(+)	320aa
O	Thioredoxin	trxA	ET527_06980	1609487..1609882(+)	131aa
O	Glutaredoxin-like protein	nrdH	ET527_01755	409364..409627(−)	87aa
O	Peptide methionine sulfoxide reductase msrA/msrB	msrAB	ET527_05320	1193250..1194242(−)	330aa
Pb	Divalent metal cation transporter mntH	mntH	ET527_07110	1640760..1642118(−)	452aa

^aPost-translational modification, protein turnover, chaperones

^bInorganic ion transport and metabolism

^cGenes on forward strand (+); genes on reverse strand (−)

Resistance to H₂O₂

As shown in Fig. 2a, the datas of lgCFU were used to select appropriate H₂O₂ concentrations for oxidative stress induction and H₂O₂ challenge. The increased exogenous levels of H₂O₂ resulted in growth defect of strain 01. The strain 01 could survive well during the 1.0 mM of H₂O₂ challenge, indicating that these levels of H₂O₂ caused no metabolic disturbances which could impact the normal growth. The growth was stopped at a critical level of 1.5 mM of H₂O₂, which indicated a non-lethal stress. Significant cell death after the 60 min treatment with 2.0 mM of H₂O₂ was found. From Fig. 2b, c, we could also find the increase of intracellular ROS concentration in strain 01 treated by 1.5 mM of H₂O₂ with time variation. Therefore, 1.5 mM of H₂O₂ was selected for the oxidative stress induction and H₂O₂ challenge.

Fig. 2.

Effect of H₂O₂ on survival and production of ROS in B. animalis subsp. lactis 01. a Survival of early exponential phase B. animalis subsp. lactis 01 over 30 min and 60 min in MRS broth medium with H₂O₂ concentration of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mM. b Representative histograms of DCFH-DA fluorescence. Black line represented control B. animalis subsp. lactis 01 cells without H₂O₂ treatment (0 min). Green line represented B. animalis subsp. lactis 01 cells treated with 1.5 mM H₂O₂ (30 min), while red line represented cells treated with 1.5 mM H₂O₂ (60 min). c The percentage of mean fluorescence intensity relative to control cells. Data presented are the mean ± SD (n = 3). Error bars represent standard deviations. Statistical significance was calculated using Holm–Sidak Student’s t test (*p < 0.05, **p < 0.01, and ***p < 0.001)

Evaluation of the expression profiles of the antioxidant-related genes during H₂O₂ stress

As shown in Fig. 3, the transcription rates of eight antioxidant-related genes in strain 01 were elevated by 1.5 mM of H₂O₂. The gene expressions at T1 were higher than those at T2 and T3, indicating that strain 01 exhibited antioxidant activity with high values by scavenging H₂O₂ within minutes. The ahpC showed 5.3-fold upregulated expression, and the expression of ahpF increased by 3.35-fold after 5 min of oxidative stress exposure (T1). The trend of other relative gene expressions (T1) in the thioredoxin system was as follows: trxB > bcp > trxA > msrAB > nrdH. The expression of mntH was upregulated by 5.46-fold after 5 min of oxidative stress exposure.

Fig. 3.

Effects of hydrogen peroxide on expressions of antioxidant-related genes in B. animalis subsp. lactis 01. The bar chart showed the relative mRNA levels of eight tested genes of cells in the presence of 1.5 mM H₂O₂ at the early exponential phase. Data presented are the mean ± SD (n = 3). Error bars represent standard deviations. Statistical significance was calculated using Holm–Sidak Student’s t test (*p < 0.05 and **p < 0.01)

AhpC is a typical peroxiredoxin which is directly regenerated by AhpF through electron transfer. The ahpC and ahpF complex, which is widely distributed in prokaryotes, is known to efficiently detoxify H₂O₂ (Poole et al. 2000; Seaver and Imlay 2001). The ahpC-overexpressing B. longum subsp. longum NCC2705 showed increased resistance to the endogenous H₂O₂ versus the control strain (Zuo et al. 2014). Other studies have shown the upregulation of ahpC in B. longum subsp. longum BBMN68 and B. animalis subsp. lactis BL-04 under oxidative challenge (Oberg et al. 2013; Xiao et al. 2011). In Salmonella typhimurium, ahpC is classified as a highly catalytic 2-Cys Prx, featuring a catalytic rate of more than 10⁷/M/s (Parsonage et al. 2008). These evidences indicate that the ensemble of ahpC and ahpF is one of the most important antioxidant proteins in strain 01.

The thioredoxin system plays a crucial role in defensing against ROS for anaerobes (Jean et al. 2004; Mishra and Imlay 2013). Besides the AhpC and AhpF complex, other antioxidant proteins may also play important roles. Bcp, a member of peroxiredoxin, exhibits hydroperoxide peroxidase activities (Jeong et al. 2000). Poorly defined in Bifidobacterium sp., bcp was characterized with antioxidant functions from Candidatus liberibacter asiaticus and Thermococcus kodakaraensis KOD1 (Singh et al. 2017; Lee et al. 2015). The methionine residues are easily oxidized by ROS. Methionine sulfoxide reductase AB (msrAB) can catalyze the methionine sulfoxides back to methionine, and the cyclic interconversion of methionine can lead to the removal of ROS (Lee et al. 2009). Strain 01 showed significant upregulation of trxB and trxA, along with nrdH. The three genes were predicted for regenerating bcp and oxidizing msrAB by electron donation in response to the oxidative stress in strain 01.

Manganese has been proven beneficial for defensing against oxidative stress, acting as a co-factor of antioxidant enzymes and non-proteinaceous manganese antioxidants (Wang et al. 2014). The acquisition of manganese is crucial for cell manganese homeostasis. MntH, a high-affinity transporter, is highly selective for manganese (Kehres and Maguire 2003). The ability to obtain manganese plays an important role in catalase-void bacteria (Turner et al. 2015). To effectively cope with oxidative stress, strain 01 enhanced manganese import through the increased expression levels of mntH.

Conclusions

Unsurprisingly, anaerobic bacteria would exhibit antioxidant activity. The antioxidant activity is important for the survival of gut microbiota in GIT filled with ROS. Strain 01 lacks the most common antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase. The antioxidant capacities of strain 01 may be associated with the thioredoxin system and manganese. Our study provides the transcriptional landscape of strain 01 under H₂O₂ challenge, and the results are highly meaningful for understanding the molecular mechanisms of ROS resistance in Bifidobacterium sp.

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Abbreviations

ROS

Reactive oxygen species

PCR

Polymerase chain reaction

RT-qPCR

Real-time quantitative PCR

COG

Clusters of orthologous groups

CFU

Colony-forming units

CDS

Coding DNA sequences

RAST

Rapid annotation using subsystem technology

GIT

Gastrointestinal tract

CICC

China Center of Industrial Culture Collection

Author contributions

JLZ and PLL designed the experiments. JLZ and SBW performed the experiments. JLZ, ZZ, SBW, and YXQ analyzed the results and wrote the manuscript. All authors read and approved the final manuscript.

Funding

This project was funded by the National Natural Science Foundation of China (31671831 and 31471707).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.