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Functional Structure/Activity Relationships
Transcriptomic analysis of xylan oligosaccharides utilization systems in Pediococcus acidilactici strain BCC-1 Zhao Lei, Yu qin Wu, Wei Nie, Dafei Yin, Xiaonan Yin, Yuming Guo, Samuel Aggrey, and Jianmin Yuan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00210 • Publication Date (Web): 22 Apr 2018 Downloaded from http://pubs.acs.org on April 22, 2018
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Journal of Agricultural and Food Chemistry
Transcriptomic analysis of xylan oligosaccharides utilization systems in Pediococcus acidilactici strain BCC-1 Zhao Lei†, Wei Nie†, Dafei Yin†, Xiaonan Yin†, Yuming Guo†, Samuel E. Aggrey§, Jianmin Yuan†,*
† State
key Laboratory of Animal Nutrition, College of Animal Science and
Technology, China Agricultural University, 2 Yuanmingyuan West Road, Beijing, 100193, PR China §NutriGenomics
Laboratory, Department of Poultry Science, University of Georgia,
Athens, GA 30602, USA
Zhao Lei:
[email protected] Wei Nie:
[email protected] Dafei Yin:
[email protected] Xiaonan Yin:
[email protected] Yuming Guo:
[email protected] Samuel E. Aggrey:
[email protected] Jianmin Yuan:
[email protected] *Corresponding author at: State key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, 2 Yuanmingyuan West Road, Beijing, 100193, PR China. Tel.:+86 010 62732337; fax: +86 010 62732712 E-mail address:
[email protected] (Jianmin yuan)
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ABSTRACT: Xylanoligosaccharides (XOS) is the hydrolysates of xylan. To
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compare the proliferation effect of XOS, glucose, fructooligosaccharides (FOS),
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xylose, XOS and a media without carbohydrate source (control) on Pediococcus
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acidilactici strain BCC-1. The de-novo sequencing of Pediococcus acidilactici strain
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BCC-1
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xylooligosaccharide between xylose and XOS was revealed through transcriptomic
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analysis. Compared to FOS, glucose and xylose, XOS exhibits a good performance
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in promoting the fermentation of Pediococcus acidilactici BCC-1. The genome of
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Pediococcus acidilactici BCC-1 revealed genes encoding XOS transportation and
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utilization related enzymes, including ATP-binding cassette (ABC) transporters,
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arabinofuranosidase, xylanase, xylosidase, xylose isomerase, and xylulose kinase.
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Transcriptome analysis showed XOS treatment enhanced genes involved
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carbohydrate metabolism, ABC transporter sugar system, pentose and glucuronate
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interconversions, pyruvate metabolism and TCA process when compared to xylose
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treatment. It suggested XOS treatment enhanced sugar absorption and utilization.
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These results are useful in the understanding of the metabolic pathway of XOS in
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Pediococcus acidilactici BBC-1 and may contribute to the optimization of the
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probiotic effect of Pediococcus acidilactici BCC-1 as novel complex feed
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additivities.
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KEYWORDS: Pediococcus acidilactici, Xylan oligosaccharides, Prebiotic effect,
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Whole genome properties, Transcriptomics
was
conducted,
and
the
underlying
mechanism
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prebiotic
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Introduction
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Probiotics, including Bifidobacterium, Lactobacillus, and Bacillus are now widely
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used for promoting health of the host. Numerous studies have confirmed the widely
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positive effect brought by probiotics, including protection against pathogenic
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bacteria,1 alleviation of allergic disease symptoms,2 and reduction of intestinal
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inflammations and immune regulation.3
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Prebiotics (mainly oligosaccharides) cannot be digested by enzymes secreted by
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the host but can be fermented by bacteria in the gut, especially the hindgut.4
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Xylanoligosaccharides (XOS) is the hydrolysates of xylan and consists of a
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backbone of xylose. The prebiotic effect of XOS has garnered much attention in
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recent years due to its fermentation properties. Compared to FOS, XOS even showed
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a better proliferation effect on Lactobacillus and Bacillus subtilis.5 Besides, XOS is
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more easy to pass through the foregut and get fermented in the hindgut due to its
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structure characteristics, which produced an efficient regulation of the hindgut
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health.6 Moreover, XOS that substituted with ferulic acid can even produce an
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anti-oxidant effect.7
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Pediococcus acidilactici belongs to Lactobacillaceae, and is one kind of
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probiotics that permitted to be used as feed additivities in many countries. Our
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previous research reported an increased number of Lactobacillaceae in the cecum of
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broilers fed a wheat-based diet supplemented with xylanase.5 We isolated a strain
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Pediococcus acidilactici BCC-1, which has the propensity to utilize XOS efficiently.
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However, the underlying molecular mechanism remains elusive.
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In this study, the Pediococcus acidilactici BCC-1 genome feature was
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investigated by de-novo sequencing. Transcriptome analysis was applied to elucidate
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the molecular mechanism utilized by Pediococcus acidilactici BCC-1 in
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interrogating XOS and other carbon sources. The underlying mechanisms may
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unearth novel ways to optimize the use of prebiotics and probiotics in the poultry
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and livestock industries.
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Materials and Method
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Materials. XOS (extracted from corncob, 95% purity, DP of 2-7 and contain 95.6%
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XOS, 2.4% xylose, 1.3% glucose and 0.5% arabinose, 0.1% raffinose) and FOS (95%
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purity) were obtained from LongLive Biotechnology (DeZhou, Shandong, China) and
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Quantum Hi-Tech Biological (JiangMen, Guangdong, China), respectively. Xylose,
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formic acid, acetic acid, propionic acid, isobutyric acid, butyrate, isovaleric acid,
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valeric acid and lactic acid were purchased from Sigma Aldrich (Shanghai, China).
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All other chemicals and solvents used in this study were of analytical grade.
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Bacteria identification and growth of Pediococcus acidilactici BCC-1. Pediococcus
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acidilactici BCC-1 was isolated from cecum of broiler aged on 36d without feeding
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any antibiotics. The strain was identified through biochemical methods and the 16Sr
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DNA sequence was performed at Beijing Sunbiotech Co., Ltd.
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The fermentation of Pediococcus acidilactici BCC-1 BCC1 was stored, resuscitated,
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and precultivated twice using MRS broth (in which the carbon source is glucose). 4
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Isolated BCC1 cells were harvested and suspended as 1% inocula into MRS medium
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containing either glucose, FOS, xylose, XOS and a control (no carbohydrate) and
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cultured at 37°C under anaerobic conditions. Aliquots of cultures were sampled at
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regular intervals, and cell growth was determined by measuring the optical density at
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600 nm (OD600).
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Preparation of RNA and DNA. Cells for RNA isolation and purification were
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harvested from triplicate cultures at estimated early mid-exponential growth phase by
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centrifugation at 4,300 X g for 10 min at 4°C. Culture purity was verified by streaking
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onto XOS agar plates. RNA extraction was performed according to instructions of
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QIAGEN 74524 kit. RNA degradation and contamination was monitored on 1%
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agarose gels. RNA concentration was measured using Qubit® RNA Assay Kit in
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Qubit® 2.0 Flurometer (Life Technologies, CA, USA). RNA purity was checked
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using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA integrity
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was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system
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(Agilent Technologies, CA, USA). DNA extraction was performed according to
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instructions of QIAGEN 51604 kit. Isolation of genomic DNA was carried out using
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SDS method. Total DNA obtained was subjected to quality control by agarose gel
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electrophoresis and quantified by Qubit. The genome of the strains was sequenced by
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Single Molecule, Real-Time (SMRT) technology. Sequencing was performed at the
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Beijing Novogene Bioinformatics Technology Co., Ltd. SMRT Analysis 2.3.0 were
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used to filter low quality reads and the filtered reads were assembled by SOAPdenovo
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(http://soap.genomics.org.cn/soapdenovo.html) to generate the complete genome, 5
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which were confirmed by PCR amplification.
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Gene prediction, annotation and protein classification. Gene prediction was
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performed on the BCC-1 genome assembly by GeneMarkS 8 with integrated model
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which combine the GeneMarkS generated (native) and Heuristic model parameters. A
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whole genome Blast
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percentage larger than 40%) was performed against 6 databases [KEGG-Kyoto
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Encyclopedia of Genes and Genomes,10 COG-Clusters of Orthologous Groups,11
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NR-Non-Redundant Protein Database databases), Swiss-Prot,12 Gene Ontology
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TrEMB.14 A whole genome Blast 9 search (E-value less than 1e-5, minimal alignment
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length percentage larger than 40%) was performed against 4 databases for
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pathogenicity and drug resistance analysis. They are PHI (Pathogen Host
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Interactions),14 VFDB (Virulence Factors of Pathogenic Bacteria),15 ARDB
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(Antibiotic Resistance Genes Database),16 CAZy ( Carbohydrate-Active Enzymes
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Database).17 Secretory proteins were detected on the genome assembly by SignalP.18
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Type I-VII secretion system related proteins were extracted from all the annotation
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results. Type III secretion system effector proteins were detected by Effective T3.19
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Secondary metabolite gene clusters were predicted by antiSMASH.20,21 All annotation
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files were further combined into one table. Genome overview was created by Circos
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Library preparation for strand-specific transcriptome sequencing. A total amount
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of 3 µg RNA per sample was used as input material for the RNA sample preparations.
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Sequencing libraries were generated using NEBNext® Ultra™ Directional RNA
9
search (E-value less than 1e-5, minimal alignment length
13
and
to show annotation information.
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Library
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recommendations and index codes were added to attribute sequences to each sample.
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Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic
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beads. For prokaryotic samples, rRNA is removed using a specialized kit that leaves
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the mRNA. Fragmentation was carried out using divalent cations under elevated
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temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand
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cDNA was synthesized using random hexamer primer and M-MuLV Reverse
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Transcriptase(RNaseH-). Second strand cDNA synthesis was subsequently performed
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using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP
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were replaced by dUTP. Remaining overhangs were converted into blunt ends via
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exonuclease/ polymerase activities. After adenylation of 3’ ends of DNA fragments,
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NEBNext Adaptor with hairpin loop structure were ligated to prepare for
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hybridization. In order to select cDNA fragments of preferentially 150~200 bp in
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length, the library fragments were purified with AMPure XP system (Beckman
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Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB，USA) was used with
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size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C
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before PCR. Then PCR was performed with Phusion HighFidelity DNA polymerase,
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Universal PCR primers and Index (X) Primer. At last, products were purified
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(AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer
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2100 system.
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Transcriptomic analysis. Clustering of the index-coded samples was performed on a
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cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia)
Prep
Kit
for
Illumina®
(NEB,
USA)
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according to the manufacturer’s instructions. After cluster generation, the library
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preparations were sequenced on an Illumina Hiseq platform and paired-end reads
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were generated. Bowtie2 program was adopted to align the reads to the genome.
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HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then
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FPKM of each gene was calculated based on the length of the gene and reads count
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mapped to this gene. Differential expression analysis of two conditions/groups (three
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biological replicates per condition) was performed using the DESeq R package
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(1.18.0). DESeq provides statistical routines for determining differential expression in
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digital gene expression data using a model based on the negative binomial distribution.
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The resulting P-values were adjusted using the Benjamini and Hochberg’s approach
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for controlling the false discovery rate. Genes with an adjusted P-value
