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Chemosphere 244 (2020) 125475

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Improving cadmium mobilization by phosphate-solubilizing bacteriavia regulating organic acids metabolism with potassium

Wan-Li Li, Jun-Feng Wang, Yao Lv, Hao-Jie Dong, Li-Li Wang, Tao He, Qu-Sheng Li*

Guangdong Key Laboratory of Environmental Pollution and Health, School of Environment, Jinan University, Guangzhou, 510632, China

h i g h l i g h t s

* Corresponding author.E-mail address: liqusheng@21cn.com (Q.-S. Li).

https://doi.org/10.1016/j.chemosphere.2019.1254750045-6535/© 2019 Elsevier Ltd. All rights reserved.

g r a p h i c a l a b s t r a c t

� Assessed organic acids metabolismand Cd mobilization of PSB affectedby Kþ.

� Kþ regulated TCA cycle via regulatingenzyme activity and related genesexpression.

� Cd mobilized by PSB increased withtartaric, fumaric and succinic acidssecretion.

� Increasing bioavailable Cd concen-trations up-regulated the biosyn-thesis of amino acids.

a r t i c l e i n f o

Article history:Received 9 September 2019Received in revised form20 November 2019Accepted 25 November 2019Available online 25 November 2019

Handling Editor: X. Cao

Keywords:Cadmium remediationGlycolysis pathwayTricarboxylic acid cycleAmino acids metabolismTranscriptomic analysis

a b s t r a c t

Organic acids secreted by phosphorus-solubilizing bacteria (PSB) is one of the main biological metabo-lites with cadmium (Cd) mobilization capacity in the conversion of insoluble precipitate forms tobioavailable forms in contaminated soil. However, the fluctuating concentrations of nutrient elementscaused by agricultural activities may result in the substantial variances of carbohydrate metabolism ofmicroorganisms involved in Cd remediation, it is therefore essential to study how metabolic strategies,especially for organic acids, affected by the environmentally friendly fertilizers, such as potassium (K). Inthis study, adding Kþ (KCl) concentrations from 0.0 to 100.0 mg/L in medium clearly accelerated Cdmobilization from 15.9 to 35.9 mg/L via inducing the secretion of tartaric acid, 3-hydroxybutyrate,fumaric and succinic acids, increased by 10.0-, 7.5-, 4.3- and 4.1-fold changes, respectively. Current datarevealed that the significant differences of metabolic pathways and genes expressions with the varied Kþ

concentrations included: ⅰ) Kþ induces a substantial up-regulation in metabolic pathway of pyruvic acidto oxaloacetate and tartaric acids; ⅱ) the varied expression of genes involved in encoding enzymes oftricarboxylic acid cycle result in the up-regulated fumaric acid, succinic acid and 3-hydroxybutyrate; ⅲ)the expression of genes related enzyme cysteine and glutamate metabolism processes promoted withthe increasing bioavailable Cd concentrations. Besides, P-type ATPase activity increased with Kþ levels,indicating that Hþ efflux and medium acidification were strengthened. In general, an appropriateenhancement of K based fertilizer is an effective manner for soil Cd remediation via the regulation oforganic acids metabolism and Hþ secretion of PSB.

© 2019 Elsevier Ltd. All rights reserved.

1. Introduction

Agricultural soil cadmium (Cd) produced from electronic

W.-L. Li et al. / Chemosphere 244 (2020) 1254752

manufacturing and disposition processes is a potentially seriousthreat to food safety (Tansel, 2017; Zhou et al., 2016). ConsideringCd usually present in an insoluble precipitate form by the combi-nationwith carbonate, iron oxide and clay (Cui et al., 2019), the bio-availability and accumulation of soil Cd would be significantlyinhibited (Hu et al., 2016). As an effective remediation technology ofsoil Cd, microorganism-plant interaction model can participate inCd mobilization from insoluble precipitate forms into bioavailableforms via secreting siderophore and organic acids (Guo et al., 2018),especially low molecular weight organic acids. However, the con-centrations of soil nutrient elements, including nitrogen (N),phosphorus (P) and potassium (K), are not always feasible forintracellular pH, osmotic solute, and enzymes activity in cells,resulting in the different concentrations of organic acids secretedby microorganisms into soil (Epstein, 2003; Forieri et al., 2017;Ruan et al., 2015). For instance, in soil with K deficiency, thebiosynthesis of jasmonic acid and genes related to nutrient trans-port and protein kinases were repressed in watermelon and riceroot, respectively (Fan et al., 2014; Zhang et al., 2017). Consequently,the metabolism and secretion of organic acids by plants will besignificantly inhibited, and the microorganism-plant system for Cdmobilization and remediation achieved at a low level (Ashley et al.,2005; Jha and Subramanian, 2016; Shen et al., 2017).

Considering the outstanding problems of aquatic environmentcaused by N and P based commercial fertilizers (Galloway et al.,2008), the application rates of these two fertilizers graduallydecreased in recent years. It is therefore essential to investigate theeffects of Kþ concentrations in soils on metabolism and secretion oforganic acids by microorganisms, and their influences on heavymetal mobilization and remediation. Organic acids are mainlyproduced in glycolysis pathway, tricarboxylic acid (TCA) cycle andother routes, such as tartaric and fatty acids metabolism (Luo et al.,2007; Yin et al., 2015). The intermediate products, including citric,malic, fumarate, succinic, and tartaric acids in TCA cycle, oxalateand tartaric acids are proposed to strengthen Cd mobilizationbecause of a good acid dissolution and complexation capacity (Weiet al., 2017). Nevertheless, the activity of enzymes related to organicacids metabolism in glycolysis pathway and TCA cycle are signifi-cantly influenced by intracellular pH, substrate concentrations, andenvironmental conditions. In fact, the common soil nutrientelement Kþ can be used to regulate the intracellular organic acidsstrategies as the activators of intracellular pyruvate kinase, gluta-mine synthase, citrate synthase, and fructose-6-phosphokinase(Hess and Haeckel, 1967; Moat et al., 2003). Meanwhile, moststudies on organic biosynthesis also indicated that this process canprovide an energy support for Kþ acquisition and organic acidssecretion (Yang et al., 2013; Yu et al., 2016). However, the varianceof enzymes activities in glycolysis pathway and TCA cycle associ-ated with different bioavailable Kþ concentrations whether or notresult in the increased production of organic acids with a better Cdmobilization ability have remained scarce.

To reveal the effects of Kþ on organic acids metabolism and Cdmobilization by microbes, Agrobacterium sp., one of phosphate-solubilizing bacteria (PSB), was selected as the model bacterium.It widely distributed in the vicinity of rhizosphere and providesnutrients such as bioavailability phosphorus for plants via secretingprotons, organic and amino acids (Chen et al., 2006; Jha andSubramanian, 2016). Of course, insoluble Cd in soil also can betransformed into bioavailable forms accompanied by the abovebiological metabolites of PSB (Jeong et al., 2013; Khan et al., 2014).As an expansion of transcriptomic analyses technology, geneticbasis underlying the physiological processes of cation binding andtransport, cellular enzymes biosynthesis, carbon and nitrogenmetabolism, and organic acids secretion have been identified(Rahman et al., 2014; Wen et al., 2015). Hence, to optimize

microorganisms for Cd mobilization, more detailed biological in-formation about the influences of Kþ on the organic acids meta-bolism and secretion are needed to be studied with metabolomicand transcriptomic analyses.

Therefore, the biological mechanisms of Cd mobilization byAgrobacterium sp. were investigated with distinct Kþ concentra-tions ranged from ~0.0 to 100.0 mg/L based on the bioavailable Kconcentrations in soils of south China (He et al., 2015; Simonssonet al., 2007). We aim to acquire that: ⅰ) changes of Cd mobiliza-tion performance affected by organic acids secreted by Agro-bacterium sp.; ⅱ) variance of organic acids strategies in glycolysisand TCA cycle, and amino acids pathways in cells; ⅲ) molecularmechanisms of Kþ in regulating expression of genes related tocation binding and transport, carbohydrate and amino acidsmetabolism, and organic acids secretion.

2. Materials and methods

2.1. Cd mobilization trial

2 mL Agrobacterium sp. solution was inoculated into a modifiedPikovskaya medium (100 mL). This medium was prepared withdeionized water and solute listed as follows (in mg/L): Glucose(1000.0), (NH4)2SO4 (500.0), Ca3(PO4)2 (668.0), NaCl (300.0), FeS-O4$7H2O (30.0), MgSO4$7H2O (534.3), MnSO4$H2O (22.7). In addi-tion, 6.0 mg CdCO3 and 30.0 mg potassium feldspar were affiliatedinto this medium as the insoluble resources of Cd and K elements,respectively. The experimental treatment was establishedwith fourKþ concentration levels of 0.0, 25.0, 50.0 and 100.0 mg/L (K0, K25,K50, and K100, respectively) with the addition of KCl. Mediumwithout Agrobacterium sp. was set up as control treatments (CK0,CK25, CK50, and CK100, respectively). Each treatment was per-formed with six replicates. After a 7-d culture period (28 �C, 180 r/min), the supernatant was obtained by centrifuging at 4000 rpm for10 min and then filtered with a 0.22 mmmicrofiltration membrane.

2.2. Determination of Cd concentration and extracellularmetabolites

OD600 value of the bacterial solution was determined withvisible spectrophotometry (UV-mini-1240, Shimadzu, Kyoto, Japan)at 1, 3, 5, 7 d periods, respectively. The pH of supernatant wasmeasured with a five easy plus pH meter (Mettler Toledo,Switzerland). K and Cd concentrations in culture solution wereanalyzed with an atomic absorption spectrometer (AA-7000, Shi-madzu, Kyoto, Japan). Intracellular pyruvate (PA) concentration andpyruvate kinase (PK) activity of strain vars were respectivelydetermined with 2,4-dinitrophenylhydrazine colorimetric methodand lactate dehydrogenase (LDH)-coupled assay, respectively (Kimet al., 2015). P-ATPase activity was analyzed in accordance with theenzyme-linked immunosorbent assays (ELISA) methods.

Extracellular metabolites in supernatant referred to the previ-ous studies (Lisec et al., 2006; Yang et al., 2018a). The dried residuewas derivatized by 20 mL methoxyamine hydrochloride (20 mg/mLin pyridine at 37 �C for 2 h) and 70 mL N-methyl-N-(trimethylsilyl)trifluoroacetamide (at 37 �C for 30 min). Samples (1 mL) wereinjected into gas chromatography-mass spectrometry (GC-MS) in asplitless mode (GC-MS-QP2010, Shimadzu, Kyoto, Japan). GC-MSdata were carried out with the automated MS deconvolution andidentification software and then compared with data in the NISTdatabase. Matching degree of >80% and retention index differenceof <20 were screened as the qualitative results of metabolites(Stein, 1999; Yang et al., 2018b). Extracellular metabolites werequantified using an internal standard method (Kumari and Parida,2018).

W.-L. Li et al. / Chemosphere 244 (2020) 125475 3

2.3. Total RNA extraction, mRNA library construction and illuminasequencing

Whole genome of Agrobacterium sp. was acquired with DNAsequencing and deposited on the NCBI database (No. CP040640-CP040642). The bacterial solution of every three medium in eachtreatment was mixed to yield one mRNA sequencing sample. TotalRNA extraction was carried out with the standard methods pro-vided by TRIzol® manufacturer after bacterial solution was frozen(�20 �C) (Rio et al., 2010). A certain amount of total RNA sampleswas settled at an appropriate temperature reaction for a while toremove DNA contaminations. Clean mRNA is collected with theapplication of rRNA purified reagent and then fragmented intosmall pieces with the fragmentation reagent. Single-strand circleDNA (cDNA) was generated using random primers reverse tran-scription, followed by double-strand cDNA synthesis. This cDNAwas subjected to end-repair and then was phosphorylated (50) andadenylated (3’). Afterward, cDNA fragments were performed withpolymerase chain reaction (PCR) amplificon. PCR product is trans-formed into a single strand, and thenwe obtained the single strandcDNA. Agilent 2100 Bioanalyzer (Agilent, Santa Clara, USA) and ABIStepOne Plus Real-Time PCR system (TaqMan Probe) were used toanalyze the quality and concentration of library, respectively. Thequalified library transformed into a strand chain by addition ofNaOH. It was diluted and then added into FlowCell and hybridizedwith the junction on FlowCell. Finally, the prepared FlowCell wassequenced by Illumina HiSeq 4000 platform in BGI company(Shenzhen, China). The raw sequencing database from RNAseq wasuploaded to the NCBI database (No. PRJNA542961).

2.4. Raw reads analysis, functional annotation and differentiallyexpressed genes identification

Raw data of reads were firstly performed with quality control(Q30 is more than 80% or Q20 is more than 90%), and then weremoved reads containing joints or with unknown base (N) above5%, and reads whose base quality value bellowed 10 or it accountedfor more than 20% of total base numbers. Clean reads werecompared with the reference whole genome with Bowtie software.Genetic expressionwas standardized by the fragments per kilobaseof exon per million fragments mapped (FPKM) method. For geneswith one more alternative transcript, the longest transcription wasselected to calculate its FPKM value.

The biological function of each gene in Gene ontology (GO),Kyoto encyclopedia of genes and genomes (KEGG), NCBI nonre-dundant protein database (NR) is obtained by BLASTX searcheswith an e-value of 10�5. To identify differentially expressed genes(DEGs) in transcriptome of Agrobacterium sp. between differentexperimental treatments, NOISeq method was used to analyzegenes expression with a reference level of FPKM value (|log1.5Ratio|>1) and probability level (>0.8). According to GO or KEGG anno-tation results, we classified DEGs into different functional cate-gories. Additionally, phyper function in R software was used toanalyze GO and KEGG enrichment.

2.5. Statistical analysis

The difference in metabolites evaluated principal componentanalysis (PCA) with Simca-P 12.0 (Bundy et al., 2010; Guo et al.,2018). Metabolites in each treatment were deposited to metab-oanalyst 3.0 platform (http://www.metaboanalyst.ca/) to annotatetheir metabolic pathway (Guo et al., 2018). One-way analysis ofvariance (ANOVA) and student’s t-tests were used to obtain thesignificant differences of pH, OD600 value and Cd mobilization

between different treatments (SPSS Ver. 22.0, IBM, USA). Statisticalanalysis of this study used p � 0.05 as significant level. R software(Ver. 3.2) was used for hierarchical cluster analysis.

3. Results and discussion

3.1. Cd mobilization by Agrobacterium sp.

The concentrations of water-soluble Cd in medium withoutstrain ranged from 7.7 to 8.1 mg/L, whereas its contents in K0, K25,K50, and K100 treatments were 15.9, 22.8, 27.9 and 35.9 mg/L,respectively (Fig. 1A). After a 7-d culture period, the pH of experi-mental treatments decreased from 6.1 (K0) to 5.5 (K100) with anincreasing Kþ concentrations. No significant difference in OD600value was found in four treatments (p > 0.05) (Fig. 1B), suggestingthat the bacterial densities in medium is similar because the po-tassium feldspar was prepared to as K resource for the normalgrowth of Agrobacterium sp. Cd mobilization increased with theenhanced concentrations of Kþ and the decline of pH values, indi-cating that Kþ could induce the mobilization of insoluble Cd via theacidification of medium. Previous studies have found that mediumacidification was mainly occurred with two routes: protonssecreted by bacteria during the Kþ absorption process or dissocia-tion of extracellular organic and amino acids (Booth et al., 2015;Harold et al., 1970; Pinu et al., 2018). Kþ is a dominant cation in thecytosol with concentrations ranged from 1600 to 8000 mg/L(Kronzucker et al., 2003). Cellular K transporters through theplasma and tonoplast membranes can maintain K balance incytosolic by the fluxes from/to soil solution (Britto and Kronzucker,2008). Increasing Kþ concentration in medium could stimulate Kþ

influx and Hþ efflux, facilitating solution acidification (Booth et al.,2015; Harold et al., 1970). As the activators for intracellular pyru-vate kinase, glutamine synthase, citrate synthase, and fructose-6-phosphokinase, Kþ promoted the activity of pyruvate kinase andthe biosynthesis of pyruvate in Agrobacterium sp. (Fig. 1D).

3.2. Effects Kþ and Cd mobilization on extracellular metabolites

Nontarget metabolomics based on GC-MS quantified 18 me-tabolites in extracellular solution (Fig. 2 and Table S1). Comparedwith K0 treatment, the concentrations of tartaric acid, 3-hydroxybutyric acid, fumaric acid, succinic acid, valine, serine,alanine, and ribose were significantly promoted, whereas theconcentrations of lactic acid, glutaric acid, galactitol, putrescine,erythritol, and glycerol were apparently decreased with anenhancive Kþ concentrations, respectively (p < 0.05). There is nosubstantial variation in contents of glyceric, lauric, terephthalic, andstearic acids with different Kþ concentrations. Based on the cor-relation analysis (Fig. 1C), the concentrations of monocarboxylic,dicarboxylic, and amino acids have a significantly positive corre-lation with water-soluble Cd contents in medium. Our previousstudy also found that dicarboxylic acids own a good Cd mobiliza-tion capacity compared with monocarboxylic and amino acids, andCd mobilization ability of each organic acid in unit concentration islisted as follows: fumaric acid > tartaric acid > succinicacid > glutaric acid > lactic acid > 3-hydroxybutyrate > proline,serine, and alanine (Wei et al., 2017). Hence, according with abovefindings, organic acids with the better Cd mobilization capacity,especially for tartaric, fumaric and succinic acids, are promotedwith increasing Kþ concentrations, inducing medium acidificationand Cdmobilization (Yuan et al., 2014). Although valine, serine, andalanine barely enabled Cd mobilization, the secretion of aminoacids in extracellular solution increased with Kþ concentrationsbecause of the detoxification effect of these acids under the higherwater-soluble Cd contents in medium (Khan et al., 2016).

Fig. 1. Variances in pH values and Cd mobilization (A), Kþ concentrations and OD600 values (B), extracellular metabolites (C), and P-type ATPase and pyruvate kinase activities (D) inthe experimental treatments. Error bars represent mean ± standard deviation.

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3.3. De novo assembly, DEGs and functional enrichment

High quality reads of the whole genome de novo assembled into5486 unigenes as the reference genome, and 5271 genomes wereannotated in database (Table S2). Total clean reads of mRNAsequencing data of 8 samples (each treatment: 2 mixture samples)approximately ranged from 21.6 Mb to 21.8 Mb. A total of 5481expressed genes of mRNA were found when compared with thewhole genome of Agrobacterium sp., and the total mapping valuewas more than 78.3% (Table S3). With an increasing Kþ concen-trations, the number of up-regulated DEGs significantly promotedwhen compared with that of down-regulated DEGs (Table 1). Bycontrast to 0.0 mg/L Kþ concentration, there were 0, 21 and 83 up-regulated and 9, 15 and 5 down-regulated DEGs in K25, K50 andK100 treatments, respectively. These results indicated that thetranscriptomic analysis was in good quality, and Kþ induced thegenes expression in Agrobacterium sp.

DEGs among four treatments were classified into 16 GO level 2terms (Fig. 3 and Table S4). Compared with K0 treatment, no sig-nificant difference was found in the enrichment GO terms in K25treatment (Table S4). In K50, GO terms of cation transport andcellular homeostasis in biological processes, transmembrane pro-teins, cation binding enzymes in cellular components, and electrontransfer and some structural molecular activity in molecular func-tions are enriched, respectively. In K100, the enrichment GO termsare partially listed as follows: ⅰ) cellular nitrogen compoundbiosynthesis, protein metabolism, amino acid, amides and macro-molecule compounds biosynthesis in biological processes; ⅱ)intracellular small ribosomal subunit, cytoplasm, ribonucleoproteincomplex, etc. in cellular components; ⅲ) N-acetyltransferase ac-tivity, mRNA binding, N-acetyl-gamma-glutamyl-phosphatereductase, and passive transmembrane transporter activity in mo-lecular functions. According to transcriptome KEGG pathway

analysis, ribosome pathway (ko03010) is significantly enrichedwith the overexpressed of related genes (p < 0.05). These resultssuggested that the expression of genes related to cation transport,the biosynthesis of organic and amino acids, and their secretion byAgrobacterium sp. were obviously different among the fourtreatments.

3.4. Effects of Kþ and Cd mobilization on the metabolism andtranscriptome of Agrobacterium sp.

The key biological mechanisms of Cd mobilization promoted byKþ in Agrobacterium sp. included Kþ induce proton efflux, organicacids strategies in glycolysis pathway and TCA, and the metabolismof glycine, serine, and threonine (GSTH) in responses to the higherwater-soluble Cd levels (Guo et al., 2018). Genes involved in Kþ

absorption and effluxes, such as Kup, Trk, Kdp systems, andglutathione-regulated K-efflux system protein, have no significantregularity (Table S5). However, the activity P-type of ATPase inAgrobacterium sp. gradually increased with Kþ contents (Fig. 1D),indicating that the Hþ efflux process was strengthened in responseto the enhancive Kþ concentrations (Beard et al., 1997). Thisresulted in medium acidification, and promoted Cd mobilizationfrom the insoluble precipitate forms (Fig. 1).

The metabolomic analysis shows that organic and amino acidsstrategies of TCA cycle, butanoate, arginine, proline, GSTH meta-bolism in Agrobacterium sp. were significantly influenced by theenhancive Kþ concentrations (Fig. 4). In K50 and K100 treatments,the activity of pyruvate kinase increased with Kþ concentrations,inducing the incremental concentrations of intracellular pyruvicacid in the glycolysis process (Fig. 1D) (Wang and Wu, 2013). Ac-cording to transcriptomic KEGG analysis (Table S5), the pathway ofpyruvic acid to oxaloacetate was also up-regulated with a signifi-cantly enhancive expression of genes involved in encoding

Fig. 2. Kþ induced different extracellular metabolites (A) and metabolic pathway (B) in Agrobacterium sp. Red and blue color value mean up or down regulation of metabolites,respectively. PK represent pyruvate kinase. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Table 1Differentially expressed transcripts (DEGs) in Agrobacterium sp. induced by different Kþ concentrations.

Kþ induced up-regulated transcripts Kþ induced down-regulated transcripts

Fold change 1.51.0-1.51.5 1.51.5-1.53.0 >1.53.0 Total Fold change 1.51.0-1.51.5 1.51.5-1.53.0 >1.53.0 Total

K25 vs. K0 / / / / K25 vs. K0 6 3 / 9K50 vs. K0 15 5 1 21 K50 vs. K0 11 3 1 15K100 vs. K0 43 33 7 83 K100 vs. K0 2 3 / 5K50 vs. K25 14 12 1 27 K50 vs. K25 1 / / 1K100 vs. K25 79 39 11 129 K100 vs. K25 1 / / 1K100 vs. K50 43 14 1 58 K100 vs. K50 10 1 / 11

W.-L. Li et al. / Chemosphere 244 (2020) 125475 5

acetolactate synthase (EC: 2.2.1.6). For fumaric and succinic acids,they are common intermediate products in TCA cycle, which uti-lized the pyruvic acid as metabolic substances. Although rare DEGsrelated to enzymes were found in TCA pathway, compared with K0and K25 treatments, a relatively minor increased (~0.7 > Log1.5Ra-tio>~0.15) expressions of genes with biological functions for thebiosynthesis of citrate synthase (EC: 2.3.3.1), pyruvate dehydroge-nase (EC: 1.2.4.1 and 2.3.1.12), isocitrate dehydrogenase (EC:

1.1.1.42), dihydrolipoamide dehydrogenase (EC: 1.8.1.4), malatedehydrogenase (EC: 1.1.1.37), and aconitate hydratase (EC: 4.2.1.3)in K50 and K100 treatments were observed. These enzymesparticipate in themetabolic processes of pyruvic acid to acetyl-CoA,oxaloacetate to citrate, citrate to isocitrate, isocitrate to oxalo-succinic acid and a-ketoglutarate, and malic acid to oxaloacetate(Alteri et al., 2009; Reisch and Elpeleg, 2007). However, genesexpression related to encoding succinate dehydrogenase (EC:

Fig. 3. Kþ induced transcripts with significantly enriched GO terms in Agrobacterium sp. of K50 vs. K0 (A), and K100 vs. K0 (B).

W.-L. Li et al. / Chemosphere 244 (2020) 1254756

1.3.5.4) showed a nonsignificant decline with the enhancement ofKþ concentrations. This enzyme can catalyze the metabolic pro-cesses of fumaric acid from/to succinic acid (Iverson, 2013), lowexpression might inhibit the biosynthesis of fumaric from/to

succinic acids, inducing the accumulation of fumaric and succinicacids in intracellular of Agrobacterium sp.

Tartaric acid is produced with the catabolism of oxaloacetate orhydroxy-fumaric acid. Genes correlated to encoding pyruvate

Fig. 4. Principal component analysis (PCA) of metabolites (A) and metabolic pathwaydifference of K100 vs. K0 (B) treatments in Agrobacterium sp.

W.-L. Li et al. / Chemosphere 244 (2020) 125475 7

carboxylase (EC: 6.4.1.1) and malate dehydrogenase (EC: 1.1.1.37)were substantially overexpressed in K50 and K100 when comparedwith that of K0 and K25 treatments, inducing an increasingbiosynthesis of oxaloacetate. As presented above, the metabolicprocess of fumaric acid to malate might be inhibited with thedown-regulated expression of correlated genes, which resulted inan accumulation of fumaric acid. The enhancive concentrations ofupstreammediators to synthetize tartaric acid are most likely to aneffective manner for the accumulation of tartaric acid in intracel-lular. Glyceric acid can be converted from the metabolism of aminoacids (glycine, serine, or threonine), glyceraldehyde, and fructose(Koivistoinen et al., 2013). It was found the genes related toencoding imidazole acetyltransferase (EC: 2.2.1.2) were signifi-cantly overexpressed under the higher extracellular Kþ conditions.This enzyme involves in the conversion of glyceraldehyde to fruc-tose (Selkov et al., 1997), inducing the enhancive upstream medi-ators to produce glyceric acid. 3-carboxy butyric acid wasmetabolized from butyric acid. However, the varied expression ofgenes correlated to the corresponding metabolic processes werenot found in transcriptomic annotated information.

Based on GO enrichment analysis, the biosynthesis of aminoacids and biological processes of ribosome, tRNA, and mRNAbinding are up-regulated in K50 and K100, indicating that thisstrain might be caught in a Cd intoxication under the higher water-

soluble Cd levels. It was found that bacteria and plants couldincrementally produce proline, serine, and alanine to chelate Cd hasbeen transported in intracellular (Khan et al., 2016; Zhou et al.,2016). The biosynthesis of proline is based on transamination ef-fect with an upstream mediator of glutamic acid (Hansen andMoran, 2011). According to KEGG analysis, the pathway of 2-oxocarboxylic acid to glutamic acid was significantly up-regulatedwith overexpressed genes related to encoding glutamate synthase(EC: 1.4.1.13 and 1.4.1.14). Additionally, genes related to encodingenzymes (glutamate 5-kinase, glutamate-5-semialdehyde dehy-drogenase, and pyrroline-5-carboxylate reductase) participate inthe biosynthesis of proline from glutamic acid showed a slight in-crease in K50 and K100 treatments. These variances in genesexpression are most likely resulted in the cumulative biosynthesisof proline in intracellular. Alanine was biosynthesized from pyru-vate or aspartic acid with an upstream mediator of oxaloacetate(Cooper et al., 2011). As showed in the TCA cycle, oxaloacetatebiosynthesis was promoted by related genes overexpressed. Thegenes correlated to encoding aspartate-semialdehyde dehydroge-nase (EC: 1.2.1.11), an enzymewith biological function to synthesizeaspartic acid, was significantly overexpressed with in K50 andK100. Moreover, the biosynthesis of serine was produced with thetransamination effect associated with an upstream mediator ofaspartic acid, inducing the incremental biosynthesis of alanine andserine. Previous studies also found that cysteine and glutamatemetabolism processes played an important role in Cd detoxificationthrough conjugating with transported Cd (Kieffer et al., 2009; Zhouet al., 2016).

Lactic acid is produced during the anaerobic respiration processof bacteria with pyruvic acid as the upstream mediator. Glutaricacid can be produced with the metabolism of 5-aminopentanoicacid during the amino acid metabolism processes, and then in-volves in biosynthesis of acetyl co-A in the TCA cycle. With the up-regulated of oxaloacetate, citric acid, a-ketoglutarate, and malicacid, the anaerobic metabolism processes and consumption ofacetyl co-A might be inhibited under the higher Kþ concentrations.Putrescine is produced by the catabolism of ornithine, agmatineand spermidine. However, genes expression data showed thatpathway of ornithine synthesis from proline was up-regulated, andthe pathway of putrescine production from ornithine was also up-regulated. In addition, the biosynthesis process of terephthalic acidin extracellular is not yet clear, the genes expression data related tothe biosynthesis of terephthalic acid was also not found in tran-scriptomic annotated information. Therefore, further researchesare required to have a depth understand of the regulation mecha-nisms of Kþ on organic and amino acids strategies of PSB.

It is found that secretion of amino acids (alanine, lysine, histi-dine) and organic acids (succinic acid, pyruvate acid) are co-transported with Hþ into an extracellular solution based on amodel organism of Escherichia coli. (Buurman et al., 2004). That is,during the efflux process of Hþ, these organic and amino acids willalso be secreted into medium through plasma membrane. Theenhancive Kþ concentrations have pointed out that the P-typeATPase activity of Agrobacterium sp. gradually increased, indicatingthat Kþ not only altered the metabolic pathways of intracellularorganic and amino acids, but also involved in the secretory path-ways related to organic acids transport via plasma membrane.However, ABC transporters with biological function of organic acidsand some amino acids did not present with a significant up or downregularity (Table S5). Genes expression related to encoding fructoseand glutamate transporter showed a slight enhancement with theincreasing Kþ concentrations, inducing the secretion of those cor-responding compounds from cells to the extracellular solution. Ingeneral, a qualified increasing Kþ concentration could promote thestrategies and exocytosis of organic and amino acids in PSB, which

W.-L. Li et al. / Chemosphere 244 (2020) 1254758

promoted Cd mobilization in medium.

4. Conclusion

An appropriately increased Kþ concentrations can promote Hþ

secretion and medium acidification via promoting P-type ATPaseactivity. Genes related to enzymes involved in the biosynthesis oftartaric acids overexpressed, and variance in the TCA cycle resultedin the enhancive fumaric and succinic acids concentrations, whichare most likely the key processes to mobilize Cd in the insolubleforms. In turn, the increasing water-soluble Cd concentrations alsostimulated GSTH metabolism to produces more amino acids todetoxicate Cd intoxication in intracellular. As a result, K based fer-tilizer at an appropriate level is one of the potential methods for Cdcontaminated soils remediation through regulation of Hþ secretionand organic and amino acids metabolism in PSB.

Notes

The authors declare no competing financial interest.

Author contributions section

Wan-Li Li, Yao Lv, Hao-Jie Dong, and Tao He performed the Cdmobilization trial in this study. Jun-Feng Wang and LieLi Wanghave written and polished this manuscript. The experimentalscheme was designed by professor Qu-Sheng Li.

Acknowledgements

This works was supported by the National Natural ScienceFoundation of China (No. 41673094) and the National Key ResearchProject of China (No. 2017YFD0801305).

Appendix A. Supplementary data

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.chemosphere.2019.125475.

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