IAI Accepted Manuscript Posted Online 22 June 2015 Infect. Immun. doi:10.1128/IAI.00312-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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ArcA Controls Metabolism, Chemotaxis and Motility Contributing to the Pathogenicity of Avian Pathogenic E. coli
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Fengwei Jiang,a,$ Chunxia An,a,$ Yinli Bao,a Xuefeng Zhao,c Robert L. Jernigan,c Andrew Lithio,d Dan Nettleton,d Ling Li,e,f Eve Syrkin Wurtele,e,f Lisa K. Nolan,b Chengping Lu,a and Ganwu Lia,b,*
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Department of Veterinary Preventive Medicine, College of Veterinary Medicine, Nanjing
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Agricultural University, Nanjing, Jiangsu 210095, P. R. Chinaa; Department of Veterinary
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Diagnostic and Production Animal Medicine, College of Veterinary Medicine, 1802 University
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Blvd., Iowa State University, Ames, Iowa 50011, USAb; Department of Molecular Biology,
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Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011, USAc; Department of
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Statistics, Iowa State University, Ames, Iowa 50011, USAd; Department of Genetics,
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Development, and Cellular Biology, Iowa State University, Ames, Iowa 50011, USAe; Center for
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Metabolic Biology, Iowa State University, Ames, Iowa 50011, USAf
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These authors contributed to this work equally.
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*
Corresponding author:
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phone: (515) 294-3358
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fax:
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e-mail:
[email protected]
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(515) 294-1401
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Avian pathogenic E. coli (APEC) cause one of the three most significant infectious diseases
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in the poultry industry and are also potential food-borne pathogens threating human
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health. In this study, we show that ArcA (Aerobic Respiratory Control), a global regulator
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important for E. coli’s adaptation from anaerobic to aerobic conditions and control of that
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bacterium’s enzymatic defenses against ROS, is involved in the virulence of APEC.
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Deletion of arcA significantly attenuates virulence of APEC in the duck model. RNA-Seq
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analyses comparing the APEC wild type and the arcA mutant indicates that ArcA regulates
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the expression of 129 genes including genes involved in citrate transport and metabolism,
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flagella synthesis, and chemotaxis. Further investigations revealed that citCEFXG
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contributed to APEC’s microaerobic growth at the lag and log phases cultured in duck
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serum and ArcA played the dual role in the control of citrate metabolism and
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transportation. In addition, deletion of flagella genes motA and motB, and chemotaxis gene
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cheA significantly attenuated the virulence of APEC and ArcA was shown to directly
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regulate the expression of motA, motB and cheA. The combined results indicate that
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ArcA controls metabolism, chemotaxis and motile contributing to the pathogenicity of
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APEC.
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KEY WORDS
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Avian pathogenic E. coli (APEC), Extraintestinal Pathogenic E. coli (ExPEC), ArcA,
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Chemotaxis, motility, and virulence.
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Commensal and pathogenic Escherichia coli can grow under both aerobic and anaerobic
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conditions. The enzymes required for catabolism under aerobic versus anaerobic conditions are
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substantially different, and E. coli have the ability to switch among anaerobic, anaerobic
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respiratory, and fermentative pathways (1). The expression of genes involved in cellular
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functions such as nutrient uptake and/or excretion systems, biosynthetic pathways, and
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macromolecular synthesis are also adjusted in response to oxygen availability (2). The Arc two-
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component signal transduction system, comprised of the kinase sensor ArcB and its cognate
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response regulator ArcA, is one of the mechanisms that enable E. coli adaptation to changing
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oxygen availability (3). Once an environmental signal is received, ArcB undergoes
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autophosphorylation and catalyzes the transphosphorylation of ArcA, which then promotes or
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represses expression of Arc-regulated genes. Rather than directly detecting environmental O2,
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ArcB probably senses the redox state of the cell through detection of a reduced electron transport
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component (4). Under aerobic conditions (5), oxidized forms of quinone electron carriers in the
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membrane inhibit the autophosphorylation of ArcB; while under anaerobic and microaerobic
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conditions, ArcB undergoes autophosphorylation. The net activity of ArcB as a kinase for ArcA
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is expected to progressively increase during transition from aerobic to anaerobic growth.
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The ArcA regulon of E. coli K-12 strains has been extensively studied under both aerobic
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and anaerobic conditions (6, 7). A previous study indicated that about 1139 genes in the E. coli
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K-12’s genome are regulated either directly or indirectly by the ArcA protein (6). Under
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anaerobic conditions ArcA inhibits the expression of genes required for aerobic metabolism,
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energy generation, amino acid transport, and fatty acid transport.
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involved in tricarboxylic acid cycle and cytochrome o ubiquinol oxidation were repressed by
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ArcA under anaerobic conditions (8). ArcA also functions as an activator. Transcription of about
In particular, the genes
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93 genes was positively regulated by ArcA in the absence of oxygen: most of these genes
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contribute to the anaerobic respiratory and metabolism pathways (6).
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A second transcription factor involved in controlling anaerobic gene expression and
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facilitating bacterial adaptation to anaerobic conditions is FNR (fumarate and nitrate respiration)
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(9). A comparison of the ArcA and FNR regulons showed that 303 genes were regulated by both
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proteins (6). Our recent study showed that FNR is a key global regulator of ExPEC virulence
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controlling expression of several important virulence traits.
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adherence and invasion; modulation of hemolysin expression; and expression of a novel
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pathogenicity island involved in α-ketoglutarate metabolism under anaerobic conditions (10).
These include motility and
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Based on the similar roles of ArcA and FNR in bacterial adaptation to anaerobic conditions,
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we propose that ArcA should also be important for pathogenic E. coli’s virulence. However, the
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regulon of ArcA in pathogenic E. coli and its role in pathogenesis have never been explored. To
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address these questions, we constructed and characterized an arcA mutant and a complemented
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strain in an avian pathogenic E. coli (APEC). The genes regulated by ArcA in duck serum were
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identified by use of RNA-Seq analyses. 129 genes were significantly differentially expressed
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between WT and arcA mutant and ArcA selectively regulated genes required for the metabolism,
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motility, and chemotaxis contributing to APEC virulence.
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MATERIALS AND METHODS
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Bacterial strains, culture conditions, and plasmids. The APEC XM (O2:K1:H7) belonging to
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the phylogenetic E. coli reference (ECOR) group B2 was isolated from the brain of a duck which
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had septicemia and neurological symptoms (11). The strain was also found to cause serious
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meningitis in a neonatal rat model of human disease. The strains and plasmids used in this study
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are listed in Table 1. Microaerobic growth was achieved by static culture at 37°C with 5%
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oxygen. For genetic manipulations, all E. coli strains were routinely grown in Luria-Bertani (LB)
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broth medium. Selective antibiotics and IPTG (isopropyl-β-d-thiogalactopyranoside) were added
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when necessary at the following concentrations: ampicillin (Amp), 100 μg · ml−1; kanamycin
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(Kan), 50 μg · ml−1; chloramphenicol (Chl), 25 μg · ml−1; and IPTG, 0.1 mM (11).
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Recombinant DNA techniques. PCR, DNA ligation, electroporation, and DNA gel
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electrophoresis were performed as described by Sambrook and Russell (12), unless otherwise
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indicated. All oligonucleotide primers were purchased from BGI Tech Solutions Co., Ltd. (BGI;
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Guangzhou, China) and are listed in Table S1 in the supplemental material. All restriction and
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DNA-modifying enzymes were purchased from TaKaRa Biotechnology (Dalian) CO., LTD. and
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used on the basis of the supplier's recommendations. Recombinant plasmids, PCR products, and
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restriction fragments were purified using TaKaRa MiniBEST plasmid purification kits or
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Agarose gel extraction kits (Shiga, Japan) as recommended by the supplier. DNA sequencing
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was performed at the DNA facility, Shanghai Sunny Biotechnology Co.,Ltd.
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Deletion mutants were constructed using the bacteriophage lambda red recombinase system
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described by Datsenko and Wanner (13-15) and primers used for mutagenesis are listed in Table
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S1. Chromosomal transcriptional arcA fusions were performed using XM strain. The
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homologous recombination constructions used PCR purified products with a selectable antibiotic
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resistance gene and 60-nt homology extensions. The mutants were confirmed by PCR and
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sequencing. For complementation, the coding sequences of genes plus their putative promoter
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regions were amplified (Primers used are listed in Table S1) from the XM strain and
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independently cloned into pGEN-MCS (16, 17) using NdeI and BamHI restriction sites.
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To construct the plasmid overproducing pET28a (+) -arcA- His6 fusion protein, a 717 bp
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fragment containing the coding region of arcA was obtained by PCR from genomic DNA using
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arcAHistag-F and arcAHistag-R (Table S1) carrying codons for 6×His and subsequently cloned
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into pET28a (+) vectors (Novagen, Madison, WI, USA) using BamHI and HindIII sites. The
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resultant plasmid contains pet28a-arcA- His6 under the control of the T7 promoter.
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RNA isolation, rRNA removing, sequencing library preparation, and sequencing. RNA
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from WT and arcA mutant of APEC XM, cultured in duck serum, and RNA from WT and arcA
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mutant, grown in LB broth, was extracted using an RNeasy minikit (Promega) with a 15 mins
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on-column DNase digestion (Promega) according to the manufacturer's instructions. Three
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biological replicates were used for each combination of genotype (WT or arcA mutant) and
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medium (duck serum or LB broth. The extracted RNA was quantified by the use of a Qubit 2.0
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spectrophotometer (Life Technologies, Carlsbad, CA) to estimate concentrations and then stored
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at −80°C until use. The cDNA libraries were constructed from 1,000 ng of total RNA and rRNA
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was removed using the MICROBExpress™ Bacterial mRNA Enrichment Kit (Life
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Technologies, Carlsbad, CA). The sequencing libraries were prepared using a TruSeq Stranded
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total RNA sample preparation kit (Illumina, San Diego, CA) following the guidelines of the
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manufacturers. Multiplex libraries were prepared using barcoded primers and a median insert
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size of 250 bp. Libraries were analyzed for size distribution using a Bioanalyzer and quantified
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by quantitative RT-PCR using a Kapa library quantification kit (Kapa Biosystems, Boston, MA),
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and relative volumes were pooled accordingly. The pooled libraries were sequenced on an
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Illumina HiSeq platform with 100-bp end reads following standard Illumina protocols at BGI.
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Data analyses. Since the genome of APEC XM was not fully sequenced, the RNA
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sequencing data were mapped to the genome sequences of APEC O1 (1) using Bowtie (18). The
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mapped reads were counted by eXpress (http://bio.math.berkeley.edu/eXpress/). Genes with an
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average of at least one uniquely mapped read across samples and at least four nonzero read
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counts were tested for differential expression between genotypes within each growth medium,
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using the R package QuasiSeq (http://cran.r-project.org/web/packages/QuasiSeq). The negative
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binomial QLShrink method implemented in the QuasiSeq package and described by (19) was
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used to compute a p-value for each gene and each genotype comparison. The log of each count
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mean was modeled as the sum of an intercept term, a genotype effect, a growth media effect, an
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interaction between genotype and growth media, and an offset normalization factor, determined
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for each sample by the log of the TMM normalization factor (20). Estimates of the fold change
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between the different growth media, and between genotypes, were computed by evaluating the
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exponential function at estimates of effect differences. Using the p-values for each comparison,
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the approach of (21) was used to estimate the number of genes with true null hypotheses among
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all genes tested, and this estimate was used to convert the p-values to q-values (22).To control
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false discovery rate at 10%, comparisons with q-values no larger than 0.10 and estimated fold
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changes of at least 2 were declared significant.
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Reverse transcription PCR and quantitative real-time RT-PCR. For the co-transcription
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test, one microgram of total RNA was reverse transcribed in triplicate using random hexamers
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and ImProm-II reverse transcriptase (Promega). Reactions without reverse transcriptase were
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used as a DNA contamination control. cDNA was then used as template for subsequent PCR
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reactions.
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Real-time quantitative reverse transcription-PCR (RT-PCR) (qPCR) was used to validate the
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expression levels for selected genes. Primers are listed in Table S1. Total RNA from APEC XM
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WT and arcA mutant, cultured in LB and serum, was extracted as described above. Treatment of
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total RNA with DNase was performed, followed by subjection to purification using the RNeasy
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kit. RNA samples without reverse transcription served as PCR templates to confirm that they
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were free of contaminating DNA. One microgram of total RNA was reverse transcribed in
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triplicate using random hexamers and Moloney murine leukemia virus (MMLV) reverse
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transcriptase (Promega). Primer pairs used are listed in Table S1, and real-time PCR was
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performed as previously described (23, 24). Melting-curve analyses were performed after each
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reaction to ensure amplification specificity. Differences (n-fold) in transcripts were calculated
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using the relative comparison method, and amplification efficacies of each primer set were
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verified as described by Schmittgen et al. (25). RNA levels were normalized using the
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housekeeping gene of the replication terminator protein Tus as a control (23, 24).
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Electrophoretic mobility shift assay. E. coli BL21 with pET28a-arcA was grown in 200
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ml of LB medium for 16 h at 28°C and protein expression was induced by adding 0.1 mM of
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IPTG. The ArcA-His6 fusion protein was purified to homogeneity using Ni-nitrilotriacetic acid
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spin columns and dialyzed in buffer (20mM Tris, 50mM NaCl, 40 mM EDTA, 4mM DTT, 10%
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glycerol, pH7.4) at 4°C for overnight and change buffer for three times. To study the binding of
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ArcA to the DNA probe, electrophoretic mobility shift assays (EMSAs) were performed as
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described previously. Briefly, DNA probes were amplified using specific primers and purified
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using a TaKaRa MiniBEST gel extraction kit. EMSAs were performed by adding increasing
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amounts of purified ArcA-His6 fusion protein (00 to 3000 ng) to the DNA probe (50 ng) in
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binding buffer (10 mM Tris-HCl, 7mM MgCl2, 50% glycerol, 40ug/ml bovine serum albumin,
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pH 8.0) for 45 min at room temperature. The reaction mixtures were then subjected to
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electrophoresis on a 6% polyacrylamide gel in 0.5× TBE buffer (44.5 mM Tris, 44.5 mM boric
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acid, 1 mM EDTA, pH 8.0) at 200 V for 45 min. The gel was stained in 0.5×TBE buffer
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containing 1× SYBR Gold nucleic acid staining solution (Life Technologies, Grand Island, NY)
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for 30 min, and then the image was recorded.
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Animal tests. Healthy 7-day-old ducks testing negative for antibodies to APEC XM were used
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in this study. All the animals were allowed free access to sterile water and animal feed and
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handled according to the guidelines of the Laboratory Animal Monitoring Committee of Jiangsu
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Province. 7-day-old ducks were divided into different groups according to our experiment,
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mortalities were recorded by intraperitoneal challenge with various doses of viable bacteria.
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Strains were grown in LB medium, at 37°C to the logarithmic phase and diluted as required in
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sterile PBS; 200μl of various doses of inoculum by intraperitoneal route. Eight ducks were used
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per dose. The ducks were observed for 7 days, and mortality rates were calculated. Fisher's exact
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tests at significance level 0.05 were used to identify statistically significant differences between
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estimated mortality rates.
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RNA-Seq data accession number. RNA-Seq data are available at the European
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Bioinformatics
Institute
ArrayExpress–functional
genomics
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(http://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-3396.
database
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RESULTS
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Deletion of arcA significantly attenuates virulence of APEC in the duck model. To address
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the role of ArcA in APEC virulence, we compared the APEC XM wild type with its isogenic
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arcA deletion mutant strain for its ability to grow in vitro (LB) under aerobic and anaerobic
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conditions. The arcA mutant grew only slightly worse than the WT under aerobic and anaerobic
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conditions (Figure S1). The virulence of WT and mutant strains were then compared using a 7-
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day- old duck model. The WT and mutant strains were inoculated into eight ducks with
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inoculation doses of 5x106, 1x106, 5x105, and 1x105 CFU. No significant difference (P>0.05) in
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mortality between WT and mutant strains was observed in the groups with higher inoculation
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doses. However, the WT strain killed significantly (P
