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Integrative genomic and proteomic analysis of response of Lactobacillus casei Zhang to glucose restriction Jie Yu, Wenyan Hui, ChenXia Cao, Lin Pan, Heping Zhang, and Wenyi Zhang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00886 • Publication Date (Web): 06 Feb 2018 Downloaded from http://pubs.acs.org on February 12, 2018
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Integrative genomic and proteomic analysis of response of Lactobacillus casei
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Zhang to glucose restriction
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Jie Yu†, §, Wenyan Hui†, §, Chenxia Cao†, §, Lin Pan†, § Heping Zhang†, §, Wenyi Zhang *†, §
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†
7
Inner Mongolia Agricultural University, Inner Mongolia, Huhhot, 010018, China.
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§
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Mongolia Agricultural University, Inner Mongolia, Huhhot, 010018, China.
Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education,
Key Laboratory of Dairy Products Processing, Ministry of Agriculture, Inner
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ABSTRACT
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Nutrient starvation is an important survival challenge for bacteria during industrial
29
production of functional foods. As next-generation sequencing technology has greatly
30
advanced, we performed proteomic and genomic analysis to investigate the response
31
of Lactobacillus casei Zhang to a glucose-restricted environment. L. casei Zhang
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strains were permitted to evolve in glucose-restricted or normal medium from a
33
common ancestor over a 3-year period, and they were sampled at 1000, 2000, 3000,
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4000, 5000, 6000, 7000, and 8000 generations and subjected to proteomic and
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genomic analyses. Genomic resequencing data revealed different point mutations and
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other mutational events in each selected generation of L. casei Zhang under glucose
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restriction stress. The differentially expressed proteins induced by glucose restriction
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were mostly related to fructose and mannose metabolism, carbohydrate metabolic
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processes, lyase activity, and amino acid-transporting ATPase activity. Integrative
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proteomic and genomic analysis revealed that the mutations protected L. casei Zhang
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against glucose starvation by regulating other cellular carbohydrate, fatty acid, and
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amino acid catabolism; phosphoenolpyruvate system pathway activation; glycogen
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synthesis; ATP consumption; pyruvate metabolism; and general stress response
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protein expression. The results help reveal the mechanisms of adapting to glucose
45
starvation and provide new strategies for enhancing the industrial utility of L.
46
casei Zhang.
47
KEY WORDS: Genomic and proteomic analysis; Glucose restriction; Lactobacillus
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casei Zhang, adaptive evolution
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INTRODUCTION
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Probiotics, including lactobacilli and bifidobacteria, are of increasing interest
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because of their health benefits, and they represent an important growth area in the
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functional food industry.1, 2 However, probiotic lactobacilli encounter various stresses
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during industrial processing. Among various environmental stresses, nutrient
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starvation represents an important survival challenge.3 This stressful environmental
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change causes growth retardation in lactobacilli or prompts them to enter the
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stationary growth phase.4 Nutrient starvation in lactobacilli may result from nutrient
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consumption for growth or indirect energy loss in some extreme environmental
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conditions.5 Nutrient starvation stress can induce probiotic lactobacilli to activate a
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comprehensive and complex stress response involving various metabolic pathways.6
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Therefore, it is necessary to study the molecular mechanism by which probiotic
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lactobacilli adapt to the stressful environmental conditions of nutrient starvation.
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Studies on the molecular mechanisms underlying adaptation to nutrient
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starvation stress generally focus on a single nutrient as a stress factor, such as carbon
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(glucose), phosphate, ornitrogen sources.7, 8 Redon et al. explored the progressive
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adaptation of Lactococcus lactis to carbon starvation using transcriptome analysis and
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revealed that 67 genes were transiently induced at the onset of carbon starvation or
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during the deceleration phase.9 In addition, some of these differentially regulated
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Lactococcus lactis genes were functionally related to glucose exhaustion, including
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the induction of the arginine deiminase pathway as well as alternative sugar transport
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and metabolic pathways.9 Recently, Butorac et al. used de novo sequencing in
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positive and negative mass spectrometry ion modes to investigate the adaptation of
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Lactobacillus brevis to nutrient deprivation and found that numerous proteins engaged
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in glucose and amino acid catabolism were differentially expressed after long-term
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starvation; however, genomic analysis was not performed in this study.10
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L. casei Zhang, a strain isolated from koumiss samples collected from Inner
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Mongolia, China, has been considered a probiotic bacterium via selection tests.11-13 A
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proteomic study to identify proteins expressed by L. casei Zhang in the exponential
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and stationary phases revealed that the differentially expressed proteins were mainly
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categorized as the key components of central and intermediary metabolism.14
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Considerable research has focused on the response of L. casei Zhang to acid stress.11,
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15-17
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L. casei Zhang in low pH conditions and highlighted the protective mechanisms of
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aspartate in the acid resistance of L. casei Zhang.15 However, little is known about the
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molecular mechanisms by which L. casei Zhang adapts to nutrient starvation.
For instance, Zhang et al. employed adaptive evolution to generate acid-resistant
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In this study, we used integrative proteomic and genomic analysis to investigate
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the response of L. casei Zhang to a glucose-restricted environment. Tandem mass tag
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(TMT)-based quantitative proteomic analysis and whole-genome resequencing were
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performed to study the adaptive evolution process over a 3-year period. The results
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may lay a solid theoretical foundation for screening stress-resistant L. casei Zhang
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and provide the necessary theoretical guidance for the optimization and improvement
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of industrial processes involving the bacterium.
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Experimental Section 4
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Experimental design
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Proteomic profiling of L. casei Zhang evolved in glucose-limited medium
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(medium L) or normal medium (medium N) was performed on day 1 and after 1000,
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2000, 3000, 4000, 5000, 6000, 7000, and 8000 generations, with each selected
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generation containing three biological replicates. The day 1 expression profiles
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(Ancestor-N-P1 or Ancestor-L-P1) were used as the baseline profiles for each evolved
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population. TMT quantitative proteomics analysis was performed using a
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high-resolution mass spectrometer. Data screening was performed with a false
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discovery rate (FDR) of 20, and reads < 50
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nucleotides length. BWA-MEM (version: 0.7.12 parameters: default parameters)22
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was
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(https://www.ncbi.nlm.nih.gov/nuccore/NC_014334.2). Variant [single nucleotide
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polymorphisms (SNPs) and insertions/deletions (indels)] calls were made using
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GATK UnifiedGenotyper and filtered using VariantFiltration in GATK with the
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settings “QD < 2.0 || FS > 60.0 || MQ < 40.0 || HaplotypeScore > 13.0 ||
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MappingQualityRankSum < -12.5 || ReadPosRankSum < -8.0"; for indel: QD < 2.0 ||
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FS > 200.0 || ReadPosRankSum < -20.0.” The variants were annotated with SnpEff23
applied
to
align
trimmed
reads
to
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to identify the genetic changes of the variants at functional level. Moreover, Cnvnator
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(version: v0.3.3; parameters, default parameters) was used for copy number variation
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(CNV) analysis. Structure variation (SV) analysis was performed using breakdancer
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(version: v1.4.5; parameters, default parameter). The SV types included deletion,
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insertion, inversion, intrachromosomal translocation, interchromosomal translocation,
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and unknown. The corresponding genes of missense mutations were subjected to
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Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation.24
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Protein extraction and digestion
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The proteomic profiles of L. casei Zhang evolved in glucose-limited medium
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(medium L) or normal medium (medium N) were determined on day 1 and after 1000,
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2000, 3000, 4000, 5000, 6000, 7000, and 8000 generations of growth. The day 1
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expression profiles (Ancestor-N-P1 or Ancestor-L-P1) were used as the baseline
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profiles for each evolved population.
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For proteomic analysis, L. casei Zhang samples were lysed using SDT lysis
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buffer (Invitrogen, Carlsbad, CA, USA). After centrifugation for 15 min at
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14,000 ×g at 25°C, the sample supernatant was collected. Next, the protein
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concentration was determined using the Pierce BCA protein Assay Kit (Thermo
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Fisher Scientific, Waltham, MA, USA).
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TMT labeling and high-pH reversed-phase peptide fractionation
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TMTs with varying molecular weights (126–131 Da) (Thermo Fisher Scientific)
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were applied as isobaric labels for the identifying the differential protein expression
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between L. casei Zhang cells cultured in medium N or medium L. L. casei Zhang 8
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strains sampled after 1000, 2000, 3000, 4000, 5000, 6000, 7000, and 8000 generations
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of growth in medium N or medium L were independently prepared three
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biological replicates per generation. The digested samples were individually labeled
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with TMT reagents according to the manufacturer’s protocols. One hundred
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microgram of sample was labeled with a TMT tag dissolved in acetonitrile. The
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labeled peptide mixtures were then fractionated using high-pH reversed-phase
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chromatography, and 10 fractions were collected. The fractions were desalted and
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lyophilized to dryness.
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LC-MS/MS
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LC-MS/MS analysis was performed using the UltiMate 3000 Nano LC System
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(Thermo Fisher Scientific) coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap
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mass spectrometer (Thermo Fisher Scientific) with a nanoelectrospray ionization
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source. The Orbitrap mass spectrometer was operated in a data-dependent mode. Each
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full MS scan (60,000 resolving power) was followed by six MS/MS scans, and the
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three most abundant molecular ions were dynamically selected and fragmented by
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collision-induced dissociation with a normalized collision energy of 35% and
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subsequently scanned by higher-energy collisional dissociation (HCD)-MS/MS with a
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collision energy of 45%, as described previously25. Only the 2+, 3+, and 4+ ions were
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selected for fragmentation by collision-induced dissociation and HCD.
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Database search, TMT quantification, and bioinformatics analysis
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An original map file (.raw file) was generated by TMT quantitative proteomics
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analysis using the high-resolution Q Exactive mass spectrometer (Thermo Scientific). 9
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The original map file was then reformatted to an .mgf file by the Proteome Discoverer
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1.4 software26 (Thermo Scientific). The data were submitted to the MASCOT2.2
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server for database retrieval via built-in software tools. Then, the library file (.Dat file)
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on the MASCOT server was submitted back to the software with Proteome
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Discoverer 1.4. Highly reliable quantitative results were obtained by data screening
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with a FDR of A,
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Cys204Tyr) with the physical location of 491277 at chromosome (located in gene
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LCAZH_RS02655, Protein ID: ADK17779.1, ABC transporter ATPase), the
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missense mutation (311C>A, Ala104Asp) with the physical location of 1343906 at
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chromosome (located in gene LCAZH_RS07010, Protein ID: ADK18290.1,
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hypothetical protein LCAZH_1047), the missense mutation (223C>A, Arg75Ser)
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with the physical location of 753319 at chromosome (located in gene
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LCAZH_RS03915, Protein ID: ADK18034.1, hypothetical protein LCAZH_0749),
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the missense mutation (382G>T, Asp128Tyr) with the physical location of 2279226
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at chromosome (located in gene LCAZH_RS11610, Protein ID: WP_043925993.1,
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peptide ABC transporter permease), the missense mutation (523G>T, Ala175Ser)
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with the physical location of 568537 at chromosome (located in gene
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LCAZH_RS02970, Protein ID: ADK19028.1, dipeptide/tripeptide permease), and the
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missense mutation (1172C>A, Ala391Asp) with the physical location of 2591574 at
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chromosome (located in gene LCAZH_RS13330, Protein ID: ADK18101.1, 13
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phosphotransferase (PTS) system galacitol transporter subunit EIIC). Moreover,
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pathway enrichment analysis revealed that the 20 mutations located to genes that were
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mainly associated with starch and sucrose metabolism, pyrimidine metabolism, purine
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metabolism, PTS system, galactose metabolism, RNA polymerase, and Glyoxylate
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and dicarboxylate metabolism.
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Competition assays and fitness trajectory
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Fitness improved among the evolved genomes over this period (Figure 2). The
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average fitness gain of L. casei Zhang under glucose restriction began as an initially
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rapid increase from generation 1000 to generation 3000 and then tended to decelerate
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from generation 3000 to generation 6000, followed by another decline from
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generation 7000 to generation 8000. However, the relative fitness of L. casei Zhang
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evolved under glucose restriction was significantly higher than that of L. casei Zhang
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cultured in medium N (pA, Arg>Ser) with the physical location of
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753319 at chromosome was actually present in 7000g and 8000g of the L group. The
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other five missense mutations, including 523G>T (Ala175Ser; chr. position: 568537),
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773T>G (Phe258Cys; chr. position: 835829), 634G>A (Val222Ile; chr. position:
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1727053), 722C>T (Pro241Leu; chr. position: 1729962), and 382G>T (Asp128Tyr;
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chr. position: 2279226) were confirmed in the generations that were consistent with
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the results of genomic re-sequencing.
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DISCUSSION
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As a probiotic bacterium, L. casei Zhang faces several challenges such as glucose
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starvation within industrial processes. To investigate the molecular mechanisms
385
employed by the bacterium to overcome glucose starvation, we performed integrative
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genomic and proteomic analysis to compare the responses of L. casei Zhang under
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glucose restriction and normal conditions.
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Evidence has revealed that sugar starvation generally triggers sequential changes
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as follows: arrest of cell growth; degradation of lipids and proteins; rapid
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consumption of cellular carbohydrate content; decrease in respiration; increase in
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accumulation of Pi, phosphorylcholine, and free amino acids; and decrease in
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glycolytic enzymatic activities.18, 19, 30, 31 The ABC transporter family is equipped with
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an extremely high substrate affinity, and it utilizes the energy of ATP binding
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and hydrolysis to drive the unidirectional accumulation of solutes across membranes
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into the bacterial cytoplasm.32 Ohtsu et al. demonstrated that the uptake of L-cysteine
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via an ABC transporter contributes to defense against oxidative stress in Escherichia
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coli.33 Another study illustrated that the differential expression of membrane transport
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system components, including ABC transporters, to aid physiological adaptations
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furfural stress in C. beijerinckii 8052.34 When subjected to glucose restriction, L. casei
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Zhang cells downregulate the expression of LCAZH_RS01795 (sugar ABC 18
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transporter periplasmic protein), which presumably protects cells against glucose
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stress by repressing glucose consumption with consumption of amino acids from the
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medium via an ABC transporter. Simultaneously, an increase in enzymatic activities
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related to the catabolism of other types of cellular carbohydrates, fatty acids, amino
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acids, and proteins occurs, including the upregulation of LCAZH_RS03175,
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LCAZH_RS03180,
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LCAZH_RS04890 (HAD superfamily hydrolase), and LCAZH_RS03270 (lactose
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transport regulator). Such a change allows protein and lipid catabolism to compensate
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for sugar catabolism and sustain respiration and metabolic processes, which is
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considered a selective advantage for survival and growth during carbon
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starvation.35Since
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acid-transporting ATPase activity, the glucose starvation condition might have
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induced L. casei Zhang to increase transport and metabolism of amino acids, which is
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also consistent with a previous study showing that the Lactococcus lactis cells
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retained the ability to transport protein substrates via ATP-driven translocation after
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carbohydrate depletion and all of the lactococci became nonculturable by inducing the
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metabolism of amino acids that resulted in ATP and new metabolic products.36
418
Notably,
LCAZH_RS04810
differentially
during
glucose
(HAD
expressed
stress,
family
proteins
sugar
are
ABC
sugar
phosphatase),
involved
transporter
in
amino
ATPase
419
(LCAZH_RS02025) and multidrug ABC transporter ATPase (LCAZH_RS10765)
420
were upregulated in L. casei Zhang of the L group. The higher ATPase activity may
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be suggestive of an increase in ATP consumption, 37,38 which has been shown in in the
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physiological regulation of nutritionally starved cells. Moreover, proteomic data also 19
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illustrated that glycogen synthase proteins regulating glycogen synthesis39 were
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differentially expressed in L. casei Zhang of the L group compared to the N group.
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Upregulation of these proteins may contribute to the synthesis of glycogen and
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subsequently help protect cells against glucose restriction. On the other hand, studies
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have shown that starved L. lactis could induce cross-protection under different stress,
428
including heat, acid, oxidative, osmotic and freezing stress.40,
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trehalose acts as an osmoprotectant as well as a carbon source in bacteria.42 In this
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study, trehalose-6-phosphate hydrolase (gene locus: LCAZH_RS13360) was
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differentially expressed in L group compared with N group. Additionally, we found a
432
missense mutation (C>A, Ala>Asp) with the physical location of 2597660 at
433
chromosome in gene LCAZH_RS13360. Moreover, this missense mutation was
434
experimentally validated and we found that the mutation was present in 7000g and
435
8000g of L. casei Zhang. In this context, it is surmised that the missense mutation
436
(C>A, Ala>Asp) in gene LCAZH_RS13360 may be assoicated with the differential
437
expression of trehalose-6-phosphate hydrolase to some extent. Moreover, the presence
438
of such proteins and mutations may be involved in the adaption to glucose deficiency
439
or cross-protection to different stress. Nevertheless, more experimental verifications
440
are required to determine whether the starvation stress in L. casei Zhang involves
441
similar factors or driven through alternative pathways.
41
The disaccharide
442
Bacterial PTS catalyzes the concomitant transport and phosphorylation of
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numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar
444
derivatives.43 Changes in carbohydrate metabolism have been observed during the 20
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response of L. casei BL23 to bile stress.44 A study demonstrated that PTS of L. casei
446
also played a role in cold shock response.45 In addition to carbohydrate PTS, most
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proteobacteria possess a paralogous system such as nitrogen PTS
448
pathway likely plays an important role in transporting periplasmic glucose into the
449
cytoplasm in addition to glucose-specific PTS.46 In this study, many proteins
450
associated with PTS were found to be upregulated, such as LCAZH_RS14555,
451
LCAZH_RS13800, LCAZH_RS13260, LCAZH_RS01875, and LCAZH_RS13410.
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This observation may reflect that another sugar other than glucose was metabolized in
453
L. casei Zhang under glucose restriction , and that the extremely slow-growing cells
454
were prepared for glucose starvation. Thus, identifying specific protein markers
455
associated with adaptation to nutrient starvation would facilitate the selection of
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strains with better performance under such stress.
46
. The PTS
457
Moreover, in this study, L. casei Zhang of the L group exhibited higher
458
expression of proteins relating to pyruvate metabolism, including LCAZH_RS07125,
459
LCAZH_RS07120, LCAZH_RS11600, LCAZH_RS09420, and LCAZH_RS09370.
460
In many microorganisms, the control of glycolytic flux depends on pyruvate kinase
461
activity.47 Pyruvate is the output of the anaerobic metabolic process known
462
as glycolysis,48 which can lead to an increase in ATP production.49 The observed
463
increase in the expression of these proteins may therefore simply reflect a need to
464
overcome glucose deficiency. In this study, some stress response proteins were also
465
induced, including the DNA mismatch repair protein MutS (LCAZH_RS11130) and
466
helicase subunit of the DNA excision repair complex (LCAZH_RS04670). Previous 21
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research has demonstrated that overrepresentation of repeats in stress response genes
468
could
469
microorganisms.50 Therefore, regulation of the expression of general stress response
470
proteins could be a common self-rescue response caused by glucose deficiency in L.
471
casei Zhang.
472
CONCLUSION
be
a
strategy
to
increase
versatility
under stressful conditions
in
473
We performed integrative genomic and proteomic analysis to study glucose
474
deficiency-induced alterations in L. casei Zhang. In response to glucose restriction
475
stress, L. casei Zhang activated a global regulatory program, and a number of changes
476
that occurred in concert to reduce the impact of glucose starvation. The integrative
477
genomic and proteomic analysis facilitated an understanding of the adaptive
478
mechanisms in L. casei Zhang during glucose restriction stress. These results will
479
prompt detailed investigations concerning nutrient starvation in L. casei Zhang and
480
may provide new strategies to increase glucose restriction stress tolerance in this
481
species of industrial importance.
482 483
ACKNOWLEDGEMENTS
484
This research was supported by the National Natural Science Foundation of China
485
(Grant No. 31601454) and the Natural Science Foundation of Inner Mongolia (Grant
486
No. 2017JQ06).
487 488
AUTHOR INFORMATION
489
Corresponding Author: Dr. Wenyi Zhang; Phone: 86-471-4316324. Fax: 22
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86-471-4305357. Email addresses:
[email protected] 491
ORCID:
492
Wenyi Zhang: 0000-0001-5530-4210
493
Jie Yu: 0000-0001-6019-9646
494
Author Contributions
495
W.Y.Z. and H.P.Z. designed the experiments. J.Y., W.Y.H. and C.X.C. performed the
496
majority of the experiments. W.Y.Z. and L.P. analyzed the data. W.Y.Z. and J.Y.
497
wrote the manuscript.
498
Notes:
499
The authors declare no competing financial interest.
500
The genomes of the evolved populations have been deposited in the National Center
501
for
502
(http://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi) under accession number SRP106668.
503
The mass spectrometry proteomics data have been deposited to the ProteomeXchange
504
Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride) with the
505
dataset identifier PXD006643.
Biotechnology
Information
(NCBI)
Sequence
Read
Archive
(SRA)
506 507
ABBREVIATIONS
508
ANOVA: analysis of variance; CFUs: colony forming units;
509
variation;
510
collisional dissociation; indels: insertions/deletions;
511
of genes and genomes;
512
phosphoenolpyruvate system; SNP: single nucleotide polymorphism;
513
nucleotide variants; SV: structure variation;
FDR: false discovery rate; GO: Gene Ontology;
CNV: copy number HCD: higher-energy
KEGG: Kyoto encyclopedia
MRS: de Mann-Rogosa-Sharpe;
PTS:
TMT: tandem mass tag.
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SNV: single
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ASSOCIATED CONTENT
515
Supporting information
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The following supporting Information files are available free of charge on the ACS
517
Publications website at DOI:
518
Details of gene mutations and differentially expressed proteins.
519
Table S1. Statistics of CNV calls.
520
Table S2. Results of SV calling.
521
Table S3. The up-regulated and downregulated proteins in L group.
522
Table S4. The up-regulated and downregulated proteins in both L and N groups.
523
Table S5. Results from integrative genomic re-sequencing and proteomic analysis.
524 525
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FIGURE LEGENDS
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Figure 1. Variants found by re-sequencing Lactobacillus casei Zhang evolved
684
under glucose restriction between generations 1000 and 8000. Specific genes are
685
denoted by gene symbols. Mutations occurred at the intergenic region are indicated in
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purple, while missense, insertion, and deletion mutations are indicated in black, green,
687
and red, respectively.
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Figure 2. Fitness improvement of Lactobacillus casei Zhang evolved in
689
glucose-limited or normal medium. The blue line is the average fitness curve of the
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low-glucose stress group, and the red line is that of the normal group. The red point in
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the lower right is the number of mutations in the normal group, and the blue point is
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that in the low-glucose stress group.
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Figure 3. Heatmap of differentially expressed proteins. A, Heatmap of 609
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overlapping proteins that exhibited differential expression in bacteria cultured in
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glucose-limited medium (L group) or normal medium (N group). B, Heatmap of the
696
expression of 31 proteins that exhibited significant differences b the N and L groups
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of the same generation. C, Heatmap of 45 proteins that exhibited no significant
698
difference among the the L groups of different generations but exhibited significant
699
differences between the L and N groups of the same generation. Each row represents
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a differentially expressed protein, while each column represents a sample.Pink and
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green bars on the upper row indicate L group and N group, respectively. Color bars on
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the lower row indicate different generations. 31
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Table 1 Primers used for validation of the randomly selected six SNVs Position
SNVs
SNV samples
Primers
Sequences (5’-3’)
Amplicon size
Temperature (°C)
(bp) 568537
753319
835829
1727053
1729962
2279226
c.523G>T|p.Ala175Ser
c.223C>A|p.Arg75Ser
c.773T>Gp.Phe258Cys
c.634G>A|p.Val222Ile
c.722C>T|p.Pro241Leu
c.382G>T|p.Asp128Tyr
5000L/6000L/7000L/8000L LAC1-F
CGCCACTACTGGTCTTTC
579
52
LAC1-R
TCAACGGATACGGATTTT
LAC2-F
CTTAGCCACCACTTATTTATTAACACC 349
58
LAC2-R
GTCACTGCCCTCGTCATCATCTT
LAC3-F
TACGAATACGACGAAGATAA
LAC3-R
GAAGACCAAAGTGGGAAT
LAC4-F
TTTGGGCTACATTTATCA
LAC4-R
GCATTTTCTTGAGCATAA
LAC5-F
CTTGGTCACCGAAATCAC
LAC5-R
CAACTGGCATACCGAAAA
5000L/6000L/7000L/8000L LAC6-F
CGGCTATGCGTATCAAGG
LAC6-R
TTATGGGACAAGGGTTTC
5000L/8000L
2000L/3000L/4000L
2000L/3000L/4000L
2000L/3000L/4000L
SNV samples indicated that the generation of samples in which the SNV was identified by genomic re-sequencing.
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51
557
49
653
51
451
50
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Table 2. Results of SNP and insertion/deletion analysis ID
SNP
indel
insert
del
1000L
10
1
0
1
1000N
4
0
0
0
2000L
22
2
0
2
2000N
18
5
3
2
3000L
19
1
1
0
3000N
17
3
2
1
4000L
23
1
1
0
4000N
25
3
2
1
5000L
32
1
0
1
5000N
15
2
0
2
6000L
24
0
0
0
6000N
35
0
0
0
7000L
40
0
0
0
7000N
28
2
2
0
8000L
51
1
0
1
8000N
36
0
0
0
SNP, single nucleotide polymorphism; indel, insertion/deletion; insert, insertion; del, deletion.
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Table 3 Twenty missense mutations identified across all biological replicates Chromos ome position 491277
Mutation
Amino
Gene
Protein-ID
Protein description
c.611G>A
p.Cys204Tyr
LCAZH_RS02655
ADK17779.1
568537
c.523G>T
p.Ala175Ser
LCAZH_RS02970
ADK17842.1
753319
c.223C>A
p.Arg75Ser
LCAZH_RS03915
ADK18034.1
835829
c.773T>G
p.Phe258Cys
LCAZH_RS04440
ADK18101.1
836038
c.564G>T
p.Met188Ile
LCAZH_RS04440
ADK18101.1
1343906
c.311C>A
p.Ala104Asp
LCAZH_RS07010
ADK18610.1
1561161
c.713C>T
p.Ala238Val
LCAZH_RS08085
ADK18825.1
1727053 1727579 1729962
c.634G>A c.138G>T c.722C>T
p.Val222Ile p.Glu46Asp p.Pro241Leu
LCAZH_RS08965 LCAZH_RS08965 LCAZH_RS08975
ADK18993.1 ADK18993.1 ADK18995.1
ABC transporter ATPase Na+/H+-dicarboxylate symporter hypothetical protein LCAZH_0749 ion Mg(2+)/Co(2+) transport protein ion Mg(2+)/Co(2+) transport protein hypothetical protein LCAZH_1377 chromosome segregation ATPase transcriptional regulator transcriptional regulator PTS system lactose/cellobiose specific subunit IIC
1764295 2248465
c.1072C>A c.263C>A
p.Leu358Met p.Ser88Tyr
LBP_RS08325 LCAZH_RS11465
ADK19490.1
2279085
c.523G>A
p.Asp175As n
LCAZH_RS11610
ADK19518.1
2279103
c.505C>T
p.Leu169Phe
LCAZH_RS11610
ADK19518.1
2279226
c.382G>T
p.Asp128Tyr
LCAZH_RS11610
ADK19518.1
2373046 2424696
c.980C>A c.1861T>C
p.Ala327Asp p.Ser621Pro
LCAZH_RS12145 LCAZH_RS12460
ADK19625.1 ADK19686.2
2538356
c.658A>C
p.Lys220Gln
LCAZH_RS13050
2591574
c.1172C>A
p.Ala391Asp
LCAZH_RS13330
WP_04392599 3.1 ADK19835.1
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FAD/FMN-containing dehydrogenase CBS domain-containing protein CBS domain-containing protein CBS domain-containing protein acyl-CoA synthetase DNA-directed RNA polymerase subunit beta' peptide ABC transporter permease PTS system galacitol transporter subunit EIIC
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Table 4. Comparison of 387 L group-specific differentially expressed proteins between the L and N groups of the same generation Number of total differential Group
Up-regulated
Down-regulated
Ancestor-L-P1 vs. N-P1
51
4
55
1000 g-L-P1 vs. N-P1
145
32
177
2000 g-L-P1 vs. N-P1
136
37
173
3000 g-L-P1 vs. N-P1
141
30
171
4000 g-L-P1 vs. N-P1
142
42
184
5000 g-L-P1 vs. N-P1
147
39
186
6000 g-L-P1 vs. N-P1
125
31
156
7000 g-L-P1 vs. N-P1
110
29
139
8000 g-L-P1 vs. N-P1
150
41
191
proteins
L group, bacteria grown in glucose-limited medium; N group, bacteria grown in normal medium; g, generation.
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Table 5. Comparison of 609 overlapping proteins identified by one-way analysis of variance between the L and N groups Number of total differential Group
Up-regulated
Down-regulated
Ancestor-L-P1 vs. N-P1
29
24
53
1000 g-L-P1 vs. N-P1
194
203
397
2000 g-L-P1 vs. N-P1
174
192
366
3000 g-L-P1 vs. N-P1
189
170
359
4000 g-L-P1 vs. N-P1
207
203
410
5000 g-L-P1 vs. N-P1
197
206
403
6000 g-L-P1 vs. N-P1
128
185
313
7000 g-L-P1 vs. N-P1
121
154
275
8000 g-L-P1 vs. N-P1
190
230
420
proteins
L group, bacteria grown in glucose-limited medium; N group, bacteria grown in normal medium; g, generation.
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Table 6. Proteins in L. casei Zhang affected by glucose restriction pos
SNPs
SNP samples
protein-DEP-samples
protein-GI
protein description
gene locus
chr1: 1320629
c.1317G>T|p.Lys
7000L/8000L
1000L/2000L/3000L/4000L/5000L/6000L/70
300438820
pyruvate kinase
LCAZH_RS06885
300438820
pyruvate kinase
LCAZH_RS06885
300439009
single-stranded DNA-specific
LCAZH_RS07840
439Asn chr1: 1320811
c.1499C>A|p.Ala
00L/8000L 5000L/6000L/7000L/8000L
500Asp chr1: 1510629
c.208G>T|p.Ala7
00L/8000L 7000L/8000L
0Ser chr1: 1729962
c.722C>T|p.Pro24
1000L/2000L/3000L/4000L/5000L/6000L/70
1000L/2000L/4000L/5000L/6000L/7000L/80 00L
2000L/3000L/4000L
exonuclease
1000L/4000L/5000L/6000L/8000L
300439229
1Leu chr1: 2221734
c.414C>A|p.Val1
c.461C>A|p.Ala1
LCAZH_RS08975
subunit IIC 5000L/6000L/7000L
38Val chr1: 2594274
PTS system lactose/cellobiose specific
1000L/2000L/3000L/4000L/5000L/6000L/70
300439695
cation transport ATPase
LCAZH_RS11325
00L/8000L 6000L
1000L/3000L/5000L/6000L/7000L/8000L
300440072
transcription regulator
LCAZH_RS13345
5000L/8000L
5000L/6000L/7000L/8000L
300440075
trehalose-6-phosphate hydrolase
LCAZH_RS13360
54Asp chr1: 2597660
c.1439C>A|p.Ala 480Asp
37
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Journal of Proteome Research
Figure 1. Variants found by re-sequencing Lactobacillus casei Zhang evolved under glucose restriction between generations 1000 and 8000. Specific genes are denoted by gene symbols. Mutations occurred at the intergenic region are indicated in purple, while missense, insertion, and deletion mutations are indicated in black, green, and red, respectively. 167x164mm (600 x 600 DPI)
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Figure 2. Fitness improvement of Lactobacillus casei Zhang evolved in glucose-limited or normal medium. The blue line is the average fitness curve of the low-glucose stress group, and the red line is that of the normal group. The red point in the lower right is the number of mutations in the normal group, and the blue point is that in the low-glucose stress group. 51x32mm (600 x 600 DPI)
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Journal of Proteome Research
Figure 3. Heatmap of differentially expressed proteins. A, Heatmap of 609 overlapping proteins that exhibited differential expression in bacteria cultured in glucose-limited medium (L group) or normal medium (N group). B, Heatmap of the expression of 31 proteins that exhibited significant differences b the N and L groups of the same generation. C, Heatmap of 45 proteins that exhibited no significant difference among the the L groups of different generations but exhibited significant differences between the L and N groups of the same generation. Each row represents a differentially expressed protein, while each column represents a sample.Pink and green bars on the upper row indicate L group and N group, respectively. Color bars on the lower row indicate different generations. 57x16mm (300 x 300 DPI)
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