Differences in acid tolerance between Bifidobacterium breve BB8 and its acid-resistant derivative B. breve BB8dpH, revealed by RNA-sequencing and physiological analysis

Anaerobe 33 (2015) 76e84

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Molecular biology, genetics and biotechnology

Differences in acid tolerance between Bifidobacterium breve BB8 and its acid-resistant derivative B. breve BB8dpH, revealed by RNA-sequencing and physiological analysis Xu Yang a, Xiaomin Hang b, Jing Tan a, Hong Yang a, * a State Key Laboratory of Microbial Metabolism, and School of Life Science & Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Rd., Shanghai 200240, PR China b Institute of Bio-medicine, Shanghai Jiao Da Onlly Company Limited, Shanghai 200233, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 September 2014 Received in revised form 18 February 2015 Accepted 25 February 2015 Available online 26 February 2015

Bifidobacteria are common inhabitants of the human gastrointestinal tract, and their application has increased dramatically in recent years due to their health-promoting effects. The ability of bifidobacteria to tolerate acidic environments is particularly important for their function as probiotics because they encounter such environments in food products and during passage through the gastrointestinal tract. In this study, we generated a derivative, Bifidobacterium breve BB8dpH, which displayed a stable, acidresistant phenotype. To investigate the possible reasons for the higher acid tolerance of B. breve BB8dpH, as compared with its parental strain B. breve BB8, a combined transcriptome and physiological approach was used to characterize differences between the two strains. An analysis of the transcriptome by RNA-sequencing indicated that the expression of 121 genes was increased by more than 2-fold, while the expression of 146 genes was reduced more than 2-fold, in B. breve BB8dpH. Validation of the RNAsequencing data using real-time quantitative PCR analysis demonstrated that the RNA-sequencing results were highly reliable. The comparison analysis, based on differentially expressed genes, suggested that the acid tolerance of B. breve BB8dpH was enhanced by regulating the expression of genes involved in carbohydrate transport and metabolism, energy production, synthesis of cell envelope components (peptidoglycan and exopolysaccharide), synthesis and transport of glutamate and glutamine, and histidine synthesis. Furthermore, an analysis of physiological data showed that B. breve BB8dpH displayed higher production of exopolysaccharide and lower Hþ-ATPase activity than B. breve BB8. The results presented here will improve our understanding of acid tolerance in bifidobacteria, and they will lead to the development of new strategies to enhance the acid tolerance of bifidobacterial strains. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Bifidobacteria Acid tolerance RNA-sequencing Physiological analysis Real-time quantitative PCR

1. Introduction Bifidobacteria are common inhabitants of the human gastrointestinal tract, constituting up to 91% of the gut microbiota in breastfed infants, and 6.9% of the gut microbiota in adults [1,2]. Several species in the genus Bifidobacterium are considered probiotics, and their presence has been associated with health-promoting effects. Therefore, some bifidobacteria have been used in functional foods, especially fermented dairy products [3]. As probiotics, it is generally believed that they must survive passage through the gastrointestinal tract and reach the distal part of the intestine in sufficient

* Corresponding author. E-mail address: [email protected] (H. Yang). http://dx.doi.org/10.1016/j.anaerobe.2015.02.005 1075-9964/© 2015 Elsevier Ltd. All rights reserved.

numbers (approximately 106e108 CFU/g) to exhibit their beneficial effects [4]. However, bifidobacteria are usually exposed to various acidic environments (e.g., the low pH of fermented dairy products in which bifidobacteria are added as probiotics, and the low pH of the stomach), which reduce their viability, thereby resulting in less than the recommended sufficient numbers reaching the intestine. Consequently, acid tolerance is recognized as a desirable property of potential probiotic bifidobacteria [5]. In addition, most bifidobacteria have a weak acid tolerance [6], which limits their application in probiotic products. As part of a strategy to improve the acid tolerance of bifidobacteria, it is necessary to fully understand their acid tolerance mechanisms. To survive in acidic environments, several response mechanisms are employed by bifidobacteria, including the

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maintenance of pH homeostasis by Hþ-ATPase, the production of NH3, and the regulation of global signaling systems and the general stress response [7e10]. These studies mainly focused on the responses of bifidobacteria to acid stress or adaptation. Nevertheless, it is not clear why different strains, including acid-resistant derivatives and their parental strains, have different acid tolerance levels. Currently, next-generation sequencing technology, e.g., RNAsequencing (RNA-seq), is a powerful tool for transcriptome profiling [11,12]. Compared with microarray methods, RNA-seq provides higher efficiency and sensitivity, and can quantify lowabundance transcripts [13]. Moreover, in terms of accuracy and precision, RNA-seq is comparable to real-time quantitative PCR (RTPCR) [14]. In the present study, a comparative analysis between B. breve BB8 and its acid-resistant derivative BB8dpH was conducted, based on transcriptome and physiological data, to explore the reasons for their different acid tolerance levels.

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RDP classifier software (version 2.2) in the GenBank database (http://www.ncbi.nlm.nih.gov/blast). 2.4. Acid tolerance assays Stationary growth phase cells were harvested by centrifugation (7600  g, 10 min), washed twice with phosphate-buffered saline with 0.05% (w/v) L-cysteine (pH 7.4), and re-suspended in fresh MRSC medium (adjusted to pH 3.2 with 6 N HCl). Aliquots of cell suspension were incubated at 37  C under anaerobic conditions, and samples were withdrawn at different times, serially diluted and plated on MRSC agar (pH 6.5) plates. Plates were incubated at 37  C for 48e72 h under anaerobic conditions, after which colony forming units (CFU) were enumerated. The experiments were performed in triplicate. 2.5. Stability evaluation of acid-resistant phenotype of the derivative

2. Materials and methods 2.1. Bacterial strains and growth conditions The bacterial strains used in this study were B. breve BB8 (GenBank Accession No. HM068368) and its acid-resistant derivative BB8dpH (China General Microbiological Culture Collection Center (CGMCC) Accession No. 8370). The d in BB8dpH stands for derivative. Strains were grown under anaerobic conditions, at 37  C, in batch cultures in de Man-Rogosa-Sharpe (MRS) [15] supplemented with 0.05% (w/v) L-cysteine (MRSC, initial pH 6.5). Stationary growth phase cells were obtained after 16 h incubation, harvested by centrifugation, and then used for all subsequent experiments. After 16 h of growth, both strains grew to similar densities (approximately 9.3 log CFU/mL) and reduced the pH of the medium to 5.2. To compare the growth rate of strains B. breve BB8 and its acid-resistant derivative BB8dpH, cultures of each strain were transferred to fresh batch MRSC medium by 1% inocula, respectively, and incubated anaerobically at 37  C. Cell growth was measured spectrophotometrically at 600 nm. 2.2. Isolation of the acid-resistant derivative Cells from an overnight culture of strain B. breve BB8, previously subcultured three times in standard conditions, were washed in phosphate-buffered saline containing 0.05% (w/v) L-cysteine (pH 7.4) and then transferred to fresh MRSC medium adjusted to pH 3.2 with 6 N HCl. These cultures were incubated at 37  C for 16 h, and then cells resistant to acidic conditions were recovered by plating on MRSC agar (pH 6.5), followed by incubation at 37  C for 3e4 days. The acid-resistant derivative B. breve BB8dpH was obtained after repeating this process many times. 2.3. Molecular identification of the acid-resistant derivative Genomic DNA of the acid-resistant derivative was extracted according to the method of Ausubel et al. [16], which was slightly modified by adding a cell disruption step with a Fast prep instrument (Thermo Fisher Scientific, USA) prior to the extraction pro0 0 cedure. Primers (5 -ATAATGCGGCCGCACGGGCGGTGTGTRC-3 and 0 0 5 -TAATAGCGGCCGCAGCMGCCGCGGTAATWC-3 ) were used to amplify the 16S rRNA partial gene (900-bp fragment length) as previously described [17]. The PCR products were purified with TaKaRa DNA fragment Purification Kit (TaKaRa Biotechnology, Dalian, China) and sequenced on an ABI-Prism 3730 automated sequencer (PE Applied Biosystems. USA). Identification of the strain was accomplished by analyzing the 16S rRNA gene sequences using

The stability of acid-resistant phenotype of the derivative B. breve BB8dpH was determined according to the following procedure. Briefly, cultures of strains BB8dpH and BB8 were transferred daily into fresh batch MRSC medium (initial pH 6.5) for 20 consecutive days by 1% inocula at each transfer, respectively. After each transfer, the cultures were incubated at 37  C for 16 h anaerobically, collected and then used to evaluate their survival in acidic conditions (pH 3.2 for 4 h) as described above. The parental strain B. breve BB8 was included as a control. 2.6. Sample preparation and RNA isolation Stationary growth phase cells were collected by centrifugation (10,000  g for 10 min at 4  C), and washed twice with phosphatebuffered saline containing 0.05% (w/v) L-cysteine (pH 7.4). The cell pellets were immediately ground into a fine powder in the presence of liquid nitrogen, and then total cellular RNA was isolated from each of the cell samples using the Easy Pure RNA Kit (TransGen Biotech, Beijing, China) according to the manufacturer's protocol. RNA quality was characterized initially on an agarose gel and NanoDrop ND 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and the further assessed by RIN (RNA Integrity Number) value (>8.0) using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). 2.7. RNA-seq and subsequent data analysis Sequencing libraries were prepared using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions. Briefly, mRNA was purified from total RNA using poly-T oligoattached magnetic beads (Life technologies, CA, USA). Fragmentation of the remaining RNA was carried out using divalent cation fragmentation buffer (New England Biolabs, Ipswich, MA, USA). First-strand cDNA was synthesized using random hexamer primers. Second-strand cDNA was synthesized using dNTPs mixture containing dUTP, DNA polymerase І and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and enzymes were removed. After adenylation of 30 ends of blunt-end DNA fragments, NEBNext adaptor oligonucleotides were ligated to cDNA fragments. In order to select cDNA fragments of preferentially 200-bp in length, the library fragments were purified with AMPure XP beads system (Beckman Coulter, Beverly, USA). The index adaptors were introduced by PCR using NEB Universal PCR Primer and index adaptor primer. The second-strand cDNA containing dUTP was digested with USER enzyme (New England

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Biolabs, Ipswich, USA). The first-strand DNA fragments with ligated adaptors on both ends were selectively enriched in a 10 cycles PCR reaction, purified (AMPure XP), and quantified using the Agilent High-Sensitivity DNA assay on the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina, CA, USA) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2000 platform and 100-bp paired-end reads were generated. The raw sequencing data were deposited in the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/) under the accession number SRP047105. Clean data were obtained from raw data by removing reads containing adapter, poly-N and low quality reads. Clean data were mapped to the reference genome of B. breve ACS-071-V-Sch8b (GenBank accession number CP002743) using Bowtie2 (version: 2.0.6). For gene expression level estimation, we performed a standard calculation of reads per kilobase of exon per million mapped reads (RPKM). Prior to differential gene expression analysis, for each sequenced library, the reads counts were adjusted by edgeR program package through one scaling normalized factor. Differentially expressed genes (DEGs) from different samples were identified according to the DEGseq R package (version 1.12.0), P < 0.005. The expression of a gene with more than 2.0-fold differences between strain BB8 and BB8dpH was considered significant. 2.8. RT-PCR analysis The cDNA was synthesized from each RNA sample (500 ng) using the PrimerScript™ RT reagent Kit with gDNA Eraser (TaKaRa Biotechnology, Dalian, China) according to the manufacturer's protocol. RT-PCR was performed using Mastercycler ep realplex system (Eppendorf, Hamburg, Germany) to confirm RNA-Seq data. The primers of selected genes were listed in Supplementary Table 1. The amplification efficiency of each primer set was determined using the standard curve. The RT-PCR mixture with a total volume of 25 mL contained 12.5 mL 2  SYBR® Premix Ex Taq™ II (TaKaRa Biotechnology, Dalian, China), 0.6 mM of the forward and reverse primers, 2 mL of cDNA template, and nuclease-free water. Thermal cycling conditions comprised of 1 cycle at 95  C for 30 s, and 40 cycles at 95  C for 5 s and 60  C for 2.5 min. The gene relative expression ratio was calculated according to 2DDCt method [18], using 16S rRNA as an endogenous control with previously described primers [19]. Individual RT-PCR reactions were carried out in triplicate for each gene.

2.10. Extraction and quantification of exopolysaccharide Exopolysaccharides (EPS) were extracted according to a modified procedure of Alp et al. [21]. Briefly, after inoculation, MRSC cultures were incubated at 37  C for 16 h under anaerobic conditions. The cultures were boiled for 15 min at 100  C, kept at room temperature for 10 min, and then treated with 17% (v/v) of 85% trichloracetic acid solution. After removal of cells and proteins by centrifugation at 10,000  g for 30 min, the EPS were precipitated with 100% ethanol. The precipitates were collected by centrifugation at 14,000  g for 30 min. Total EPS (expressed as mg/L) were determined in each sample by the phenol-sulfuric acid method [22] using glucose as standard [23]. The experiments were performed in triplicate and mean values were calculated. 2.11. Statistical analysis Student's t test was employed to investigate statistical differences. Differences with P-value less than 0.05 were considered statistically significant. 3. Results 3.1. Isolation and characterization of the acid-resistant derivative The acid-resistant derivative B. breve BB8dpH was successfully isolated from its parental strain B. breve BB8 by serial exposure to pH 3.2. A partial 16S rRNA gene of the acid-resistant derivative was amplified and sequenced, and found to have 100% identity with its parental strain. To investigate the acid tolerance of B. breve BB8 and B. breve BB8dpH, the viability of stationary growth phase cells treated at pH 3.2 was determined. BB8dpH exhibited higher viability than BB8 (approximately 104e105-fold higher) after exposure to pH 3.2 for 4 h (Fig. 1). The stability of the acid-resistant phenotype of B. breve BB8dpH was also confirmed by the method described in Section 2.5. As shown in Fig. 2, the viable cell counts of BB8dpH were obviously higher than those of BB8 at every day, which indicates that a constitutive, acid-resistant derivative was obtained. Both strains grew equally well in batch MRSC medium (Supplementary Fig. 1). 3.2. Analysis and verification of RNA-seq data DEGs between strains BB8 and BB8dpH were identified using high-throughput RNA-seq. In strains BB8 and BB8dpH, 91.76% of the

2.9. Measurement of Hþ-ATPase activity Stationary growth phase cells were collected by centrifugation (10,000  g for 10 min), and then washed twice with phosphatebuffered saline containing 0.05% (w/v) L-cysteine (pH 7.4). The cell pellet was immediately ground into a fine powder in the presence of liquid nitrogen. After thawing, cells were re-suspended in phosphate-buffered saline containing 0.05% L-cysteine (w/v) (pH 7.4). Cell debris was removed by centrifugation at 4  C (10,000  g for 15 min). The protein concentration was determined by the Bradford assay using bovine serum albumin as a standard [20]. The activity of the Hþ-ATPase in cell extracts was measured using the Hþ-ATPase assay kit (Genmed Scientifics Inc., Wilmington, DE, USA) following the manufacturer's protocol. The activity of the HþATPase was expressed in micromoles of NADH oxidized per minute per milligram of protein. The experiments were repeated three times and mean values were calculated.

Fig. 1. Survival of B. breve BB8 and B. breve BB8dpH during an acid challenge (pH 3.2). Symbols: B. breve BB8 (C); B. breve BB8dpH (-). The error bars represent the standard deviations of the mean.

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involved in carbohydrate transport and metabolism (COG G), amino acid transport and metabolism (COG E), and translation, ribosomal structure, and biogenesis (COG J). There were 20 DEGs that showed no COG. 3.3. DEGs involved in carbohydrate transport, metabolism, and energy production, and Hþ-ATPase activity of BB8 and BB8dpH

Fig. 2. Analysis of the stability of the acid-resistant phenotype of B. breve BB8dpH. Cultures of strains BB8dpH and BB8 were daily transferred into fresh batch MRSC medium (initial pH 6.5) for 20 consecutive days, respectively. After each transfer, the cultures of stationary growth phase (16 h of growth, about pH 5.2) were daily collected, and then used to evaluate the survival before and after exposure to pH 3.2 for 4 h. Symbols: Strain BB8 were exposed to pH 3.2 for 0 h ( ); Strain BB8 were exposed to pH 3.2 for 4 h (B); Strain BB8dpH were exposed to pH 3.2 for 0 h (;); Strain BB8dpH were exposed to pH 3.2 for 4 h (A). The error bars represent the standard deviations of the mean.

▫

total reads (~21 million) and 79.68% of the total reads (~10 million), respectively, from the raw RNA-seq data were mapped uniquely to the genome, and only 0.98% and 3.49% of the total reads, respectively, were mapped multiply to the genome (Supplementary Table 2). These uniquely mapped reads were then used in the following analyses. Using a 2.0-fold change as a cutoff, there were 267 genes that were differentially expressed between BB8 and BB8dpH. The expression of 121 DEGs was higher and the expression of 146 DEGs was lower in BB8dpH than in BB8. To validate the results obtained from RNA-seq, the expression of 27 selected DEGs was measured using RT-PCR. As shown in Fig. 3, there was a strong positive correlation (R2 ¼ 0.91) between the RNA-seq data and the RT-PCR data. Of the 267 DEGs, 247 DEGs were grouped into functional categories according to the cluster of orthologous groups (COG) classification system. They were involved in 16 groups with specific biological functions, 1 general function prediction only (COG R) and 1 function unknown (COG S) (Fig. 4). Among the 16 groups with specific biological functions, the top three groups of DEGs were

The expression of ten DEGs, except for HMPREF9228_1985, encoding sugar ATP-binding cassette (ABC) transport system proteins was higher in BB8dpH than in BB8 (Table 1 and Fig. 3). As indicated by the Transporter Classification Database (www.tcdb. org), the substrates of ABC transport system proteins encoded by these genes include various carbohydrates (glucose, arabinose, xylose, glycogen, etc.). Nine genes related to the glycolytic pathway were differentially expressed between BB8 and BB8dpH (Table 2 and Fig. 3). Specifically, the expression of HMPREF9228_0420, tal, gap, pgk, and eno, which are involved in the conversion of glucose into phosphoenolpyruvate, was lower in BB8dpH than in BB8. Meanwhile, BB8dpH displayed a lower expression of HMPREF9228_0596, which is implicated in the formation of lactate from pyruvate, and a higher expression of adh, and HMPREF9228_0582, which are involved in the conversion of pyruvate into acetate, when compared with BB8. In addition, the expression of nox, which encodes NADH oxidase and is related to carbohydrate metabolism, was higher in BB8dpH than in BB8. Surprisingly, the expression of four DEGs (atpA, atpD, atpG, and atpH), encoding four out of eight Hþ-ATPase subunits, was lower in BB8dpH than in BB8 (Table 3 and Fig. 3). Moreover, the Hþ-ATPase activity in BB8dpH (0.06 ± 0.01 mmol NADH/min/mg protein) was significantly lower than that in BB8 (0.10 ± 0.01 mmol NADH/min/ mg protein) (P < 0.05). 3.4. DEGs involved in cell envelope components, and EPS production of BB8 and BB8dpH Peptidoglycan, EPS, and undecaprenyl-PP (UND-PP) are important components of the bifidobacterial cell envelope. Ten genes related to the cell envelope were differentially expressed between BB8 and BB8dpH (Table 4 and Fig. 3). The expression of HMPREF9228_0606, HMPREF9228_0341, HMPREF9228_1952, and HMPREF9228_1869, which are related to peptidoglycan synthesis, was higher in BB8dpH than in BB8. In regard to UND-PP synthesis, the expression of ispF and uppS was higher in BB8dpH than in BB8. In regard to EPS synthesis pathway, strain BB8dpH displayed higher expression of HMPREF9228_0453, and HMPREF9228_0454, which encode glycosyltransferases, when compared with BB8. Furthermore, the expression of dexB and glgX_1, which are involved in the conversion of glycogen to glucose, was higher in BB8dpH than in BB8. In addition, the production of EPS in BB8dpH (102.35 ± 3.06 mg/L) was significantly higher than that in BB8 (79.21 ± 2.03 mg/L) (P < 0.05). 3.5. DEGs involved in amino acid transport and synthesis

Fig. 3. Correlation of the fold change values from RNA-seq and RT-PCR. The bestefit curve is shown along with the calculated equation. The R2 value is 0.91. The values of RNA-seq fold change and RT-PCR fold change for the 27 selected genes were shown in Supplementary Table 1.

Fourteen genes involved in transport and synthesis of amino acids were differentially expressed between strain BB8 and BB8dpH (Table 5 and Fig. 3). The hisE and HMPREF9228_0588, which are involved in histidine synthesis, and gltA, acnA, glnA_1, glnH, glnQ, and HMPREF9228_0924, which are involved in glutamate and glutamine synthesis or transport, were expressed at higher levels in BB8dpH than in BB8. Conversely, ilvC_1, ilvD, ilvE, leuA, HMPREF9228_1413, and HMPREF9228_1524, which are involved in the transport or synthesis of branched chain amino acid (BCAA),

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Fig. 4. Differentially expressed genes between BB8 and BB8dpH, as categorized by functional classification according to the clusters of orthologous groups (COG) annotation. Genes included those whose expression was higher (black bars) and lower (gray bars), in BB8dpH compared to BB8.

Table 1 Differentially expressed genes involved in sugar ATP-binding cassette (ABC) transport system between BB8 and BB8dpH. Gene ID

Fold change (BB8dpH vs BB8)

COG

Code

Gene description

HMPREF9228_0355 HMPREF9228_0728 HMPREF9228_1639 HMPREF9228_1640 HMPREF9228_1870 HMPREF9228_1871 HMPREF9228_1872 HMPREF9228_1936 HMPREF9228_1937 HMPREF9228_1985

2.62 2.58 2.48 2.70 2.25 2.79 7.73 2.28 2.27 2.30

G G G G G G G G G G

COG1653 COG0395 COG0395 COG1175

Multiple sugar transport system substrate-binding protein Multiple sugar transport system permease protein Multiple sugar transport system permease protein Multiple sugar transport system permease protein Simple sugar transport system permease protein Simple sugar transport system permease protein Simple sugar transport system ATP-binding protein Raffinose/stachyose/melibiose transport system permease protein Raffinose/stachyose/melibiose transport system permease protein Multiple sugar transport system ATP-binding protein

COG1172 COG1129 COG0395 COG1175 COG3839

including valine, leucine and isoleucine, were expressed at lower levels in BB8dpH than in BB8. Table 2 Differentially expressed genes associated with glycolytic pathway between BB8 and BB8dpH. Gene ID

Fold change COG Code (BB8dpH vs BB8)

4. Discussion

Gene Gene description

HMPREF9228_0420 3.28

G

COG0166

HMPREF9228_0860 2.62 HMPREF9228_0638 4.07

G G

COG0176 tal COG0057 gap

HMPREF9228_0868 2.68

G

COG0126 pgk

HMPREF9228_1135 5.14 HMPREF9228_0596 3.68

G C

COG0148 eno COG0039

HMPREF9228_1699

2.70

C

COG1012 adh

HMPREF9228_0582 HMPREF9228_1839

2.03 3.40

C R

COG1254 COG0446 nox

Glucose-6-phosphate isomerase Transaldolase Glyceraldehyde 3-phosphate dehydrogenase Phosphoglycerate kinase Enolase L-lactate dehydrogenase Aldehyde-alcohol dehydrogenase 2 Acylphosphatase NADH oxidase

Acid tolerance is an important property that enables bifidobacteria to survive in acidic environments. However, the molecular basis for the acid tolerance of bifidobacteria is not fully understood, partly due to the paucity of efficient molecular genetics tools for the analysis of gene functions [10,24,25]. Therefore, in the present study, we used transcriptome and physiological approaches to investigate the possible reasons why the acid-resistant derivative of B. breve BB8dpH had higher acid tolerance than its parental strain B. breve BB8. The data obtained from this study allowed us to propose a model for the possible reasons for higher acid tolerance of B. breve BB8dpH. As shown in Fig. 5, the model consisted of several parts, including sugar ABC transporter systems, glycolysis, cell envelope, amino acid transporter systems, glutamine and glutamate synthesis, BCAA synthesis, and histidine synthesis. The model is discussed in detail in the following paragraphs.

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Table 3 Differentially expressed genes encoding four out of eight subunits of Hþ-ATPase between BB8 and BB8dpH. Gene ID

Fold change (BB8dpH vs BB8)

COG

Code

Gene

Gene description

HMPREF9228_0325 HMPREF9228_0326 HMPREF9228_0327 HMPREF9228_0328

3.09 4.40 3.04 2.01

C C C C

COG0712 COG0056 COG0224 COG0055

atpH atpA atpG atpD

F-type F-type F-type F-type

Hþ-transporting Hþ-transporting Hþ-transporting Hþ-transporting

ATPase ATPase ATPase ATPase

subunit subunit subunit subunit

delta alpha gamma beta

Table 4 Differentially expressed genes involved in synthesis of cell envelope components between BB8 and BB8dpH. Gene ID

Fold change (BB8dpH vs BB8)

COG

Code

HMPREF9228_0606 HMPREF9228_0341 HMPREF9228_1952 HMPREF9228_1869 HMPREF9228_1110 HMPREF9228_1206 HMPREF9228_0453 HMPREF9228_0454 HMPREF9228_1954 HMPREF9228_1094

2.52 2.35 2.00 2.76 2.72 2.07 2.27 2.67 2.57 2.05

M M K MG I I M M G G

COG0768 COG1181 COG0846 COG0245 COG0020 COG0438 COG0438 COG0366 COG1523

Gene

Gene description

ispF uppS

Cell division protein FtsI (penicillin-binding protein 3) D-alanine-D-alanine ligase NAD-dependent deacetylase 6-phospho-3-hexuloisomerase 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase Undecaprenyl diphosphate synthase Glycosyltransferase, group 1 family protein Glycosyltransferase, group 1 family protein Oligo-1,6-glucosidase Glycogen debranching protein GlgX

dexB glgX_1

Table 5 Differentially expressed genes encoding proteins involved in amino acid transport and synthesis between BB8 and BB8dpH. Gene ID

Fold change (BB8dpH vs BB8)

HMPREF9228_0816 HMPREF9228_0588

2.77 3.08

HMPREF9228_1508 HMPREF9228_0679 HMPREF9228_0590 HMPREF9228_0924 HMPREF9228_0524 HMPREF9228_1373 HMPREF9228_0107 HMPREF9228_0474 HMPREF9228_0935 HMPREF9228_0185 HMPREF9228_1413 HMPREF9228_1524

2.15 2.83 3.92 2.32 2.05 3.17 2.18 2.10 4.20 6.18 3.08 2.94

COG

Code

Gene

Gene description

E E

COG0140 COG0106

hisE

C C E RE TE E HE GE HE E CE E

COG0372 COG1048 COG0174 COG0493 COG0834 COG1126 COG0059 COG0129 COG0115 COG0119 COG0473 COG4177

gltA acnA glnA_1

Phosphoribosyl-ATP pyrophosphohydrolase Phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase; Phosphoribosylanthranilate isomerase Citrate synthase Aconitate hydratase Glutamine synthetase Glutamate synthase (NADPH/NADH) small chain ABC transporter substrate-binding protein Glutamine ABC transporter, ATP-binding protein GlnQ Ketol-acid reductoisomerase Dihydroxy-acid dehydratase Branched-chain amino acid aminotransferase 2-isopropylmalate synthase 3-isopropylmalate dehydrogenase Branched-chain amino acid transport system permease protein

4.1. DEGs involved in carbohydrate transport, metabolism, and energy production, and Hþ-ATPase activity of BB8 and BB8dpH Bacteria can use carbohydrates as carbon sources to produce energy. The sugar ABC transporters can transport carbohydrate across cellular membranes [26]. Bifidobacteria have a high carbohydrate catabolic diversity and can utilize many carbohydrates as carbon sources [27]. Previous studies reported that enhancing the ability to transport and utilize carbohydrates as carbon sources could help cells withstand acidic environments in Lactobacillus casei and Bifidobacterium longum subsp. longum BBMN68 [7,28]. In the present study, the DEGs related to sugar ABC transporter systems were involved in the uptake of various carbohydrates such as glucose, arabinose, xylose and glycogen. These carbohydrates can be used as glycolysis substrates (Part B in Fig. 5). Therefore, the higher expression of DEGs encoding sugar ABC transporters (Part A in Fig. 5) might contribute to the increased uptake of various carbohydrates and consequently provide sufficient substrates for glycolysis, which could contribute to the tolerance of BB8dpH toward adverse environments, including acidic environments. The lactate is produced from pyruvate by NADH-dependent

glnH glnQ ilvC_1 ilvD ilvE leuA

lactate dehydrogenase. The higher expression of nox gene encoding NADH oxidase (Part B in Fig. 5) might reduce the lactate production, as NADH oxidase competes with lactate dehydrogenase for NADH [29]. Moreover, transcriptional analysis of DEGs related to pyruvate catabolism (Part B in Fig. 5) also implied the reduction of lactate production and increase of acetate production, resulting in a higher acetate/lactate ratio in BB8dpH. The displacement of the bifid shunt toward acetate production could increase ATP production at the substrate level [30], and this could enhance the ability of cells to regulate the internal pH under acidic environments [28,31,32]. Therefore, the higher acid tolerance of BB8dpH might be related to the regulation of the expression of DEGs involved in ac
nchez et al. [31] reetate and lactate production. Furthermore, Sa ported that the acetic/lactic acid ratios in Bifidobacterium bifidum and Bifidobacterium infantis, were higher in bile-resistant derivatives compared with their corresponding parental strains. Considering that bile acids, the main component of bile, are weak acids and can decrease intracellular pH [24,27,33], acid tolerance is thought to have a relationship with bile tolerance. Some lactic acid bacteria, including Streptococcus and Lactobacillus, can utilize the Hþ-ATPase to resist acidic environments

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Fig. 5. A model for acid tolerance of B. breve BB8dpH. Genes with increased and decreased expression in BB8dpH compared to BB8 are shown in red and blue, respectively. The numbers displayed represent the last four digits of the locus tag (HMPREF9228_XXXX) for the genes. Abbreviations: EPS, exopolysaccharide; BCAA, branched-chain amino acid; GlnH, ABC transporter substrate-binding protein; GlnQ, glutamine ABC transporter, ATP-binding protein GlnQ; ClcN-6P, D-glucosamine-6-phosphate; DexB, oligo-1,6-glucosidase; GlgX_1, glycogen debranching protein GlgX; Fru-6P, fructose-6-phosphate; Tal, transaldolase; GA-3-P, glyceraldehydes-3-phosphate; Gap, glyceraldehyde-3-phosphate dehydrogenase; Pgk, phosphoglycerate kinase; Eno, Enolase; PEP, phosphoenolpyruvate; Pyk, pyruvate kinase; PflB, formate C-acetyltransferase; Adh, aldehyde-alcohol dehydrogenase 2; AckA, acetate kinase; Nox, NADH oxidase; IspF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; UppS, undecaprenyl diphosphate synthase; GlnA_1, glutamine synthetase; AcnA, aconitate hydratase; GltA, citrate synthase; IlvC_1, ketol-acid reductoisomerase; IlvD, dihydroxy-acid dehydratase; IlvE, branched-chain amino acid aminotransferase; LeuA, 2-isopropylmalate synthase; PRPP, phosphoribosyl pyrophosphate; HisE, phosphoribosyl-ATP diphosphatase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

[34,35], but some others, such as L. casei ATCC 344 [36], Lactobacillus reuteri [37], and Streptococcus sobrinus [38], do not. For bifidobacteria, the expression of the Hþ-ATPase subunit genes, as well as the amount of the corresponding proteins, were increased under acidic conditions in B. longum [7,10] and Bifidobacterium lactis [8]. In contrast, the production of Hþ-ATPase subunits showed no significant differences at neutral pH between an acid-resistant derivative and its parental strain in B. longum biotype longum NCIMB 8809 [10]. In another report, no differences in Hþ-ATPase activity were found between strain B. longum BL1 and its acid-sensitive derivative BL1-S in free and pH-controlled cultures [39]. In the present study, the expression of four DEGs encoding four Hþ-ATPase subunits (Table 3 and Fig. 3), as well as Hþ-ATPase activity, were lower in BB8dpH than in BB8. The reason for these discrepancies is unclear, but it is likely that the role of the Hþ-ATPase in acid tolerance is strain-specific. 4.2. DEGs involved in the synthesis of cell envelope components (peptidoglycan and EPS), and EPS production of BB8 and BB8dpH Fructose-6-phosphate can be produced from D-arabino-hex-3ulose 6-phosphate, and converted to phosphoenolpyruvate and the peptidoglycan precursor D-glucosamine-6-phosphate. The

fructose-6-phosphate toward peptidoglycan synthesis might be strengthened by increasing the expression of DEGs responsible for D-glucosamine-6-phosphate production and the conversion of Darabino-hex-3-ulose 6-phosphate to fructose-6-phosphate, and decreasing the expression of DEGs involved in conversion of fructose-6-phosphate to phosphoenolpyruvate (Part B and C in Fig. 5). Meanwhile, the higher expression of DEGs related to the production of peptidoglycan and its precursor D-alanyl-D-alanine (Part C in Fig. 5) might contribute to peptidoglycan synthesis. In addition, UND-PP is the lipid carrier that is involved in the synthesis of cell wall peptidoglycan [40]. Taken together, transcriptional analysis of DEGs related to synthesis of peptidoglycan and UND-PP (Part B and C in Fig. 5) might imply the higher production of peptidoglycan as an important component of cell wall, and consequently strengthen cell wall structure. Wu et al. [28] reported that strengthening cell wall structure could help cells protect against acidic environments. Thus, the higher expression of DEGs involved in peptidoglycan synthesis could be one of the reasons for the higher acid tolerance of BB8dpH. Glucose is a main component of EPS and participates in EPS synthesis [41,42]. Glucose can be produced from glycogen and converted to phosphoenolpyruvate in glycolytic pathway (Part B in Fig. 5). In addition, the glycosyltransferases, which catalyze the

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transfer of a monosaccharide unit from an activated sugar phosphate to an acceptor molecule, are also involved in EPS synthesis [43]. The transcriptional analysis of DEGs related to glycosyltransferases, the production and consumption of glucose (Part B and C in Fig. 5) suggested that the EPS synthesis might be strengthened in BB8dpH. Indeed, the higher production of EPS was observed in BB8dpH. Alp et al. [21] demonstrated that EPS could aid in the tolerance of bifidobacteria to acid stress. Taken together, this suggests that the higher production of EPS in BB8dpH could be one of the reasons that strain BB8dpH had the higher acid tolerance than BB8. The BCAA are precursors for iso- and anteiso-branched-chain fatty acids (BCFA) synthesis. Previous studies demonstrated that low amount of BCAA could decrease BCFA production in Lactococcus lactis subsp. cremoris [44]. Moreover, lower BCFA content could lead to lower membrane fluidity [45], which contributed to the enhancement of acid tolerance in L. casei [36] and Escherichia coli [46]. Therefore, the lower expression of DEGs involved in the synthesis and transport of BCAA (Part D and F in Fig. 5) might lead to the reduction of BCFA production and consequently decrease membrane fluidity, which could contribute to the higher acid tolerance of BB8dpH. 4.3. DEGs involved in the synthesis and transport of glutamate, glutamine, and histidine synthesis Glutamate and glutamine play a central role in nitrogen metabolism and can serve as nitrogen sources for bacteria [47]. Generally, about 88% of cellular nitrogen in bacteria comes from glutamate, and the rest comes from glutamine [48]. Therefore, the higher expression of DEGs involved in the synthesis and transport of glutamate and glutamine (Part D and E in Fig. 5) might increase the production of intracellular glutamate and glutamine, and consequently provide sufficient nitrogen sources for BB8dpH cells against acidic environments. Previous studies in L. casei ATCC 344 and B. longum subsp. longum BBMN68 reported that acid-adapted cells with higher acid tolerances displayed higher expression of genes responsible for histidine synthesis than non acid-adapted cells [7,36]. Furthermore, the addition of histidine to the medium enhanced the acid tolerance of L. casei ATCC 344 [36]. Therefore, the higher expression of DEGs related to synthesis of histidine (Part G in Fig. 5), which was thought to contribute to intracellular buffering capacity [36], might contribute to the higher acid tolerance of BB8dpH. 5. Conclusions The transcriptome and physiological data presented in this work revealed the possible reasons why the acid-resistant derivative B. breve BB8dpH had the higher acid tolerance than its parental strain BB8. The results suggested that the acid tolerance of BB8dpH was enhanced, possibly by regulating physiological processes and the expression of genes at several levels. First, strain BB8dpH might enhance the ability of cell envelope to prevent the entry of Hþ into cells by the higher production of peptidoglycan and EPS, and lower production of BCAA (BCFA precursors). Second, when the Hþ entered the cytoplasm, the BB8dpH cells could have several strategies to withstand acidic environments, including the higher acetate/lactate ratio, increment in uptake of various carbohydrates, higher production of intracellular glutamate and glutamine, and enhancement of histidine synthesis. Moreover, other DEGs were found (Supplementary Table 3) in addition to the DEGs mentioned in the discussion section of this study. So far, the role of the other DEGs in acid tolerance is unclear. Nevertheless, some of the other DEGs were found to be associated with protein synthesis and

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peptide uptake, which may reflect the physiological needs for the BB8dpH cells against acidic environments. Overall, the results presented here could contribute to the understanding of acid tolerance in bifidobacteria and the development of new strategies to enhance the acid tolerance of bifidobacterial strains. Acknowledgments This work was supported by the National Basic Research Program of China (973 Program) (No. 2012CB720802), and the National High Technology Research and Development Program of China (863 Program) (No. 2011AA100901). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.anaerobe.2015.02.005. References [1] P.S. Langendijk, F. Schut, G.J. Jansen, G.R. Raangs, M.H. Kamphuis, G.W. Welling, Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples, Appl. Environ. Microbiol. 61 (1995) 3069e3075. [2] H.J.M. Harmsen, A.C.M. WildeboereVeloo, G.C. Raangs, A.A. Wagendorp, N. Klijn, J.G. Bindels, et al., Analysis of intestinal flora development in breastfed and formula-fed infants by using molecular identification and detection methods, J. Pediatr. Gastroenterol. Nutr. 30 (2000) 61e67. [3] FAO/WHO, Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria, Rep. a Jt. FAO/WHO Expert Consult. Eval. Health Nutr. Prop. Probiotics Food Incl. Powder Milk Live Lactic Acid Bact. (2001) 1e33. Available at: ftp://ftp.fao.org/docrep/fao/009/a0512e/a0512e00. pdf (accessed 19.11.04). [4] P. Marteau, J.C. Rambaud, Potential of using lactic acid bacteria for therapy and immunomodulation in man, FEMS Microbiol. Rev. 12 (1993) 207e220. [5] S. Parvez, K.A. Malik, S. Ah Kang, H.Y. Kim, Probiotics and their fermented food products are beneficial for health, J. Appl. Microbiol. 100 (2006) 1171e1185. [6] M. Matsumoto, H. Ohishi, Y. Benno, Hþ-ATPase activity in Bifidobacterium with special reference to acid tolerance, Int. J. Food Microbiol. 93 (2004) 109e113. [7] J. Jin, B. Zhang, H. Guo, J. Cui, L. Jiang, S. Song, et al., Mechanism analysis of acid tolerance response of Bifidobacterium longum subsp. longum BBMN 68 by gene expression profile using RNA-sequencing, PloS One 7 (2012) e50777. [8] M. Ventura, C. Canchaya, D. van Sinderen, G.F. Fitzgerald, R. Zink, Bifidobacterium lactis DSM 10140: identification of the atp (atpBEFHAGDC) operon and analysis of its genetic structure, characteristics, and phylogeny, Appl. Environ. Microbiol. 70 (2004) 3110e3121. [9] L. Waddington, T. Cyr, M. Hefford, L.T. Hansen, M. Kalmokoff, Understanding the acid tolerance response of bifidobacteria, J. Appl. Microbiol. 108 (2010) 1408e1420.
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