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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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Journal of Proteome Research

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Integrative genomic and proteomic analysis of response of Lactobacillus casei

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Zhang to glucose restriction

3 4

Jie Yu†, §, Wenyan Hui†, §, Chenxia Cao†, §, Lin Pan†, § Heping Zhang†, §, Wenyi Zhang *†, §

5 6

†

7

Inner Mongolia Agricultural University, Inner Mongolia, Huhhot, 010018, China.

8

§

9

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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ACS Paragon Plus Environment

Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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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

32

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,

34

4000, 5000, 6000, 7000, and 8000 generations and subjected to proteomic and

35

genomic analyses. Genomic resequencing data revealed different point mutations and

36

other mutational events in each selected generation of L. casei Zhang under glucose

37

restriction stress. The differentially expressed proteins induced by glucose restriction

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were mostly related to fructose and mannose metabolism, carbohydrate metabolic

39

processes, lyase activity, and amino acid-transporting ATPase activity. Integrative

40

proteomic and genomic analysis revealed that the mutations protected L. casei Zhang

41

against glucose starvation by regulating other cellular carbohydrate, fatty acid, and

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amino acid catabolism; phosphoenolpyruvate system pathway activation; glycogen

43

synthesis; ATP consumption; pyruvate metabolism; and general stress response

44

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

48

casei Zhang, adaptive evolution

49 50 2


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INTRODUCTION

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Probiotics, including lactobacilli and bifidobacteria, are of increasing interest

53

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

69

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

71

the induction of the arginine deiminase pathway as well as alternative sugar transport

3



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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

75

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

88

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

90

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

92

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

103

discovery rate (FDR) of 20, and reads < 50

152

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

7


the

reference

genome


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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,

164

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

172

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

177

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

181

between L. casei Zhang cells cultured in medium N or medium L. L. casei Zhang 8


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182

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

192

(Thermo Fisher Scientific) coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap

193

mass spectrometer (Thermo Fisher Scientific) with a nanoelectrospray ionization

194

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

203

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

205

1.4 software26 (Thermo Scientific). The data were submitted to the MASCOT2.2

206

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

208

Discoverer 1.4. Highly reliable quantitative results were obtained by data screening

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with a FDR of A,

278

Cys204Tyr) with the physical location of 491277 at chromosome (located in gene

279

LCAZH_RS02655, Protein ID: ADK17779.1, ABC transporter ATPase), the

280

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

286

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)

288

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



292

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

295

metabolism, PTS system, galactose metabolism, RNA polymerase, and Glyoxylate

296

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

375

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;

17



379

chr. position: 2279226) were confirmed in the generations that were consistent with

380

the results of genomic re-sequencing.

381 382

DISCUSSION

383

As a probiotic bacterium, L. casei Zhang faces several challenges such as glucose

384

starvation within industrial processes. To investigate the molecular mechanisms

385

employed by the bacterium to overcome glucose starvation, we performed integrative

386

genomic and proteomic analysis to compare the responses of L. casei Zhang under

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glucose restriction and normal conditions.

388

Evidence has revealed that sugar starvation generally triggers sequential changes

389

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

391

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

394

and hydrolysis to drive the unidirectional accumulation of solutes across membranes

395

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

397

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

400

Zhang cells downregulate the expression of LCAZH_RS01795 (sugar ABC 18


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401

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

403

medium via an ABC transporter. Simultaneously, an increase in enzymatic activities

404

related to the catabolism of other types of cellular carbohydrates, fatty acids, amino

405

acids, and proteins occurs, including the upregulation of LCAZH_RS03175,

406

LCAZH_RS03180,

407

LCAZH_RS04890 (HAD superfamily hydrolase), and LCAZH_RS03270 (lactose

408

transport regulator). Such a change allows protein and lipid catabolism to compensate

409

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

411

starvation.35Since

412

acid-transporting ATPase activity, the glucose starvation condition might have

413

induced L. casei Zhang to increase transport and metabolism of amino acids, which is

414

also consistent with a previous study showing that the Lactococcus lactis cells

415

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

417

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

421

be suggestive of an increase in ATP consumption, 37,38 which has been shown in in the

422

physiological regulation of nutritionally starved cells. Moreover, proteomic data also 19



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423

illustrated that glycogen synthase proteins regulating glycogen synthesis39 were

424

differentially expressed in L. casei Zhang of the L group compared to the N group.

425

Upregulation of these proteins may contribute to the synthesis of glycogen and

426

subsequently help protect cells against glucose restriction. On the other hand, studies

427

have shown that starved L. lactis could induce cross-protection under different stress,

428

including heat, acid, oxidative, osmotic and freezing stress.40,

429

trehalose acts as an osmoprotectant as well as a carbon source in bacteria.42 In this

430

study, trehalose-6-phosphate hydrolase (gene locus: LCAZH_RS13360) was

431

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

443

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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445

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

447

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.

452

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

456

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



Page 22 of 41

467

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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490

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.

23


SNV: single


Page 24 of 41

514

ASSOCIATED CONTENT

515

Supporting information

516

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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526

1. Demers-Mathieu, V.; St-Gelais, D.; Audy, J.; Laurin, É.; Fliss, I. Effect of the

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paracasei/casei; and Lactobacillus plantarum isolates. J. Funct. Foods 2016, 24,

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327-337.

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cheese

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viability

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2. Corcoran, B.; Stanton, C.; Fitzgerald, G.; Ross, R. Survival of probiotic

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lactobacilli in acidic environments is enhanced in the presence of metabolizable

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sugars. Appl. Environ. Microbiol. 2005, 71, 3060-3067.

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3. van de Guchte, M.; Serror, P.; Chervaux, C.; Smokvina, T.; Ehrlich, S. D.;

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Maguin, E. Stress responses in lactic acid bacteria. Anton. Van Leeuwenhoek

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2002, 82, 187-216.

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response genes: a strategy to increase versatility under stressful conditions?

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Nucleic Acids Res. 2002, 30, 1886.

681 682

FIGURE LEGENDS

683

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

686

purple, while missense, insertion, and deletion mutations are indicated in black, green,

687

and red, respectively.

688

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

690

low-glucose stress group, and the red line is that of the normal group. The red point in

691

the lower right is the number of mutations in the normal group, and the blue point is

692

that in the low-glucose stress group.

693

Figure 3. Heatmap of differentially expressed proteins. A, Heatmap of 609

694

overlapping proteins that exhibited differential expression in bacteria cultured in

695

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

697

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

700

a differentially expressed protein, while each column represents a sample.Pink and

701

green bars on the upper row indicate L group and N group, respectively. Color bars on

702

the lower row indicate different generations. 31


Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 32 of 41

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.

32


636

51

557

49

653

51

451

50

Page 33 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.

33



Page 34 of 41

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

34


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

Page 35 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.

35



Page 36 of 41

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.

36


Page 37 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


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


Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

For TOC only

38


Page 38 of 41

Page 39 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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)



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)


Page 40 of 41

Page 41 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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)


Integrative Genomic and Proteomic Analysis of the ... - ACS Publications

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