Recently, miR-451 as a tumor suppressor has been reported in other studies. However, whether miR-451 can affect the sensitivity of non-small cell lung cancer (NSCLC) cells to cisplatin (DDP) remains unclear. The aim of this study is to evaluate the roles of miR-451 in the sensitivity of NSCLC cells to DDP.Quantitative RT-PCR assay was performed to detect the expression of miR-451 in 10 pairs of NSCLC and noncancerous tissue samples. pcDNA-GW/EmGFP-miR-451 was stably transfected into NSCLC cell line (A549). Then, the effects of miR-451 upregulation on growth, colony formation and apoptosis of A549 cells were investigated. Finally, the effects of miR-451 upregulation on in vitro and in vivo sensitivity of A549 cells of DDP were also determined.The level of miR-451 expression in NSCLC tissues was significantly higher than that in corresponding noncancerous tissues. Ectopic overexpression of miR-451 could significantly inhibit growth and induce apoptosis of A549 cells. Moreover, ectopic overexpression of miR-451 could sensitize A549 cells to DDP possibly by increasing DDP-induced apoptosis which might be associated with the inactivation of Akt signaling pathway.This study demonstrated for the first time that combination of DDP application with miR-451 upregulation might be a potential strategy for the treatment of human NSCLC. NSCLC accounts for the majority of lung cancer cases and chemotherapy has been the mainstay of treatments of lung cancers . Up to dMicroRNAs (miRNAs) are small, endogenous non-coding RNAs that have been identified as post-transcriptional regulators of gene expression. MiRNAs exert their functions through imperfect base-pairing with the 3'-untranslated region (3'-UTR) of target mRNAs -6. MeanwIn the present study, we identify miR-451 to be downregulated in human NSCLC and report for the first time that upregulation of miR-451 can enhance DDP chemosensitivity in NSCLC cell line (A549) by inducing apoptosis enhancement, which identifies miR-451 as a valid therapeutic target in strategies employing novel multimodality therapy for patients with NSCLC.A total of 10 pairs of matched NSCLC and noncancerous tissue samples were surgically obtained from patients in Nanjing Chest Hospital, Jisnsu Province and diagnosed by an independent pathologist. None of the patients had received chemotherapy or radiotherapy before surgery. Samples were snap-frozen in liquid nitrogen and stored at -80°C until RNA extraction. Written informed consent was obtained from all patients before surgery.2 with humidity at 37°C.NSCLC cell line (A549) was cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cell lines were cultured under the atmosphere of 5% COTGCTGAAACCGTTACCATTACTGAGTTGTTTTGGCCACTGACTGA- CAACTCAGTTGGTAACGGTTT-3') and miR-451-precursor-R (5'-CCTGAAACCGTTACCAAC-TGAGTTGTCAGTCAGTGGCCAAAACAACTCAGTAATGGTAACGGTTTC-3') was digested with BamHI and BglII and cloned into the BamHI-BglII fragment of the pcDNA-GW/EmGFP-miR vector . A construct including the non-specific miR-NC (99 bp) was used as a negative control. The constructed vectors were named pcDNA-GW/EmGFP-miR-451 and pcDNA-GW/EmGFP-miR-NC, respectively.The precursor sequence of miR-451 generated by annealing and primer extension with miR-451-precursor-F (5'-A549 cells were seeded into 6-well plates and transfected with the miR-415-expressing vector or the control vector expressing a non-specific miR-NC using Lipofectamine 2000 (Invitrogen), and were selected with spectinomycin (100 μg/ml) to generate two stable monoclonal cell lines .CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAAA-CTCAG-3' and U6: 5'-TGGTGTCGTGGAGTCG-3') quantified with a TaqMan probe, and normalized by U6 small nuclear RNA using TaqMan miRNA assays .Total RNA was extracted using TRIzol reagent . Reverse-transcribed complementary DNA was synthesized with the Prime-Script RT reagent Kit . Realtime polymerase chain reaction (PCR) was performed with SYBR Premix Ex Taq . For miRNA detection, mature miR-451 was reverse-transcribed with specific RT primers .Total RNA was extracted using TRIzol reagent . Reverse-transcribed complementary DNA was synthesized with the Prime-Script RT reagent Kit . Conventional PCR was used to assay miRNA expression with the specific forward primers and the universal reverse primer complementary to the anchor primer. U6 was used as internal control . The PCR primers for mature miR-451 or U6 were designed as follows: miR-451 sense, 5'-Thirty micrograms of protein extract were separated in a 15% SDS-polyacrylamide gel and electrophoretically transferred onto a PDVF membrane . Membranes were blocked overnight with 5% non-fat dried milk and incubated for 2 h with antibodies to phospharylated Akt (pAkt-473), total Akt, Bcl-2 and Bax  and GAPDH . After washing with TBST , the membranes were incubated for 1 h with horseradish peroxidase-linked goat-anti-rabbit antibody. The membranes were washed again with TBST, and the proteins were visualized using ECL chemiluminescence and exposed to x-ray film.3 cells/well) and allowed to attach overnight. After cellular adhesion, freshly prepared anticancer drugs (DDP) were added with various concentrations. After 72 h, cell viability was assessed using MTT assay. The absorbance at 490 nm (A490) of each well was read on a spectrophotometer. Three independent experiments were performed in quadruplicate.The mock or stably transfected A549 cells were seeded into 96-well plates (6.0 × 10Approximately 500 mock A549 or stable transfect A549 cells (A549/miR-451 and A549/miR-NC) were placed in a fresh 6-well plate with or without DDP for another 12 h and maintained in RMPI 1640 containing 10% FBS for 2 weeks. Colonies were fixed with methanol and stained with 0.1% crystal violet in 20% methanol for 15 min.Cells were treated with or without DDP for another 12 h and harvested and fixed with 2.5% glutaraldehyde for 30 minutes. After routine embedment and section, the cells were observed under electronic microscope. The apoptosis rates were determined using Annexin V-FITC and PI staining flow cytometry.Cells were cultured on 6-well tissue culture plates to confluence and treated with or without DDP for another 12 h. Then, Hoechst 33342  was added to the culture medium of living cells; changes in nuclear morphology were detected by fluorescence microscopy using a filter for Hoechst 33342 (365 nm). The percentages of Hoechst-positive nuclei per optical field (at least 50 fields) were counted.2 incubator. The activities were quantified spectrophotometrically at a wavelength of 405 nm.The activity of Caspase-3 was measured using Caspase-3 Colorimetric Assay Kit  following the manufacturer's instruction. In brief, cells were seeded in the 6-wells and were cultured for 24 h. Then, the cells were administered with or without DDP for another 12 h and harvested, resuspended in 50 μL of lysis buffer and incubated on ice for 30 min, and cellular debris was pelleted. The lysates (50 μL) were transferred to 96-well plates. The lysates were added to 50 μL 2.0 × Reaction Buffer along with 5 μL Caspase-3 Substrate and incubated for 4 h at 37°C, 5% COTissues were plated on polylysine-coated slides, fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 1 h at 25°C, rinsed with 0.1 M PBS, pH 7.4, and permeabilized with 1% Triton X-100 in 0.01 M citrate buffer (pH 6.0). DNA fragmentation was detected using TUNEL Apoptosis Detection Kit , which specifically labeled 3'-hydroxyl termini of DNA strand breaks using fluorescein isothiocyanate (FITC)-conjugated dUTP. DNA was also labeled with FITC DNA-binding dye for 5 min. FITC labels were observed with a fluorescence microscope. The percentage of apoptotic cells was calculated as the number of apoptotic cells per number of total cells × 100%.2 . All mice were killed and s.c. tumors were resected and fixed in 10% PBS. TUNEL staining assay was performed on 5 μm sections of the excised tumors. The number of apoptotic cells in five random high-power fields was counted.All experimental procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals and were performed according to the institutional ethical guidelines for animal experiment. Each aliquot of mock or stably transfected A549 cells were injected into the flanks of BALB/c nude mice  which were purchased from the Experimental Animal Centre of Nanjing Medical University and maintained under pathogen-free conditions (n = 8/group). One day after tumor cell implantation, mice were treated with CDDP , Tumor volume was followed up for 4 weeks and measured once weekly. The tumor volume formed was calculated by the following formula: V = 0.4 × D × dt test. Statistical significance was accepted at P < 0.05.All experimental data were shown as the mean ± SEM. Differences between samples were analyzed using the Student's In this study, a stem-loop qRT-PCR assay was performed to determine the expression of miR-451 in 10 pairs of matched NSCLC and noncancerous lung tissue samples. As shown in Figure P < 0.05). The gel electrophoresis of RT-PCR products confirmed the upregulation of miR-451 expression in A549/miR-451 cells , pcDNA-GW/miR-451 was transfected and stable transfectants (A549/miR-451 or A549/miR-NC) were successfully established. As shown in Figure s Figure .P < 0.05). The number of colonies formed from A549/miR-451 cells was significantly lower than that formed from mock A549 or A549/miR-NC cells  of DDP for 12 h or 5 μg/ml of DDP for 0, 12, 24, 26 and 48 h. The results from MTT assay indicated that upregulation of miR-451 led to a significant decrease in cell viability of A549 cells in response to DDP in a dose- or time -dependent manner compared with those of A549/miR-NC and mock A549 cells Figure  and 5B. P < 0.05). However, the apoptotic rate of A549/miR-NC cells treated with DDP showed no significant difference compared with that of mock A549 cells treated with DDP (P > 0.05). Figure P < 0.05). Therefore, upregulation of miR-451 might increase DDP chemosensitivity of A549 cells by enhancing DDP-induced apoptosis.The precise underlying mechanisms by which upregulation of miR-451 enhances chemosensitivity of A549 cells to DDP were further investigated. Then, the apoptosis was detected by flow cytometric assay. As shown in Figure 3) was significantly lower than that of A549/miR-NC (323 ± 13 mm3) following DDP treatment  that regulate gene expression by targeting mRNAs for translational repression, degradation, or both. Increasing evidence suggests that deregulation of miRNAs has been frequently observed in tumor tissues. These miRNAs have regulatory roles in the pathogenesis of cancer in humans, through the suppression of genes involved in cell proliferation, differentiation, apoptosis, metastasis and resistance -18. Rece3K/AKT signaling pathway [Here, we showed that miR-451 is frequently downregulated in human NSCLC tissues compared with corresponding noncancerous lung tissues, which is consistent with the results of Gao'et al . It was  pathway . Thus, m pathway . At the  pathway . P-glyco pathway . Olga an pathway . All theIn the present study, we found that the upregulation of miR-451 could significantly inhibit growth and colony formation of NSCLC cell line (A549). Upregulation of miR-451 could also enhance caspase-3-dependent apoptosis of A549 cells by inactivating the Akt signalling pathway which induced the reverse of Bcl-2/Bax ratio. Furthermore, upregulation of miR-451 could significantly increase the in vitro and in vivo sensitivity of A549 cells to DDP. To the best of our knowledge, we provided the first insight into the roles and possible mechanisms of miR-451 upregulation in chemosensitivity of A549 cells to DDP. These data suggest that appropriate combination of DDP application with miR-451 regulation might be a potential approach to NSCLC therapy. For higher-dose DDP would produce potentially serious toxic effects such as nephro- and ototoxicity would be increased, combination of DDP application with miR-451 upregulation for the treatment of NSCLC would contribute to lower-dose DDP administration and result in a reduction of DDP toxic side-effects. Although inhibition of Akt signal pathway has been reported to be able to improve chemotherapeutic effect of human tumor cells, whether upregulation of miR-451 enhance DDP chemosensitivity of A549 cells by inactivating the Akt signal pathway needs to be further elucidated. Moreover, only A549 cell line has been used in this study, further researches should be conducted on other cell lines to testify our experimental data.In conclusion, upregulation of miR-451 could increase the sensitivity of A549 cells to DDP both in vitro and in vivo, suggesting that appropriate combination of DDP application with miR-451 upregulation might be a potential strategy for the treatment of human NSCLC in future.The authors declare that they have no competing interests.HBB and XP contributed to clinical data, samples collection, MTT, apoptosis and caspase-3 activity detection analyses and manuscript writing. JSY contributed to animal experiment. ZXW and WD were responsible for the study design and manuscript writing. All authors read and approved the final manuscript.


Stackebrandtia nassauensis Labeda and Kroppenstedt (2005) is the type species of the genus Stackebrandtia, and a member of the actinobacterial family Glycomycetaceae. Stackebrandtia currently contains two species, which are differentiated from Glycomyces spp. by cellular fatty acid and menaquinone composition. Strain LLR-40K-21T is Gram-positive, aerobic, and nonmotile, with a branched substrate mycelium and on some media an aerial mycelium. The strain was originally isolated from a soil sample collected from a road side in Nassau, Bahamas. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of the actinobacterial suborder Glycomycineae. The 6,841,557 bp long single replicon genome with its 6487 protein-coding and 53 RNA genes is part of the  Genomic  Encyclopedia of  Bacteria and  Archaea  project. T (=DSM 44728 = NRRL B-16338 = JCM 14905) is the type strain of Stackebrandtia nassauensis, which is the type species of the genus Stackebrandtia [S. nassauensis was originally isolated by M. P. Lechevalier and subsequently described by Labeda and Kroppenstedt [Glycomyces strains. The genus was named after Erko Stackebrandt, a German microbiologist of note, who has contributed significantly to the molecular systematics the Actinobacteria. At present the genus Stackebrandtia contains only one additional species: S. albiflava, isolated from a soil sample collected from a tropical rainforest in China [S. nassauensis strain LLR-40K-21T together with the description of the complete genomic sequencing and annotation.Strain LLR-40K-21S. nassauensis. With 95% sequence similarity, the type strain S. albiflava, YIM 45751 [et al., unpublished), represents the only related phylotype (with the same degree of sequence similarity as YIM 45751). Curiously, the type strains of the neighboring genus Glycomyces [G. harbinensis [S. nassauensis (as of July 2009).A search of GenBank revealed no 16S rRNA reference sequences that were closely related to IM 45751 , is the ycomyces  were notbinensis  sharing S. nassauensis in a 16S rRNA based tree. The two 16S rRNA gene sequences in the genome of strain LLR-40K-21T are identical and do not differ from the previously published 16S rRNA sequence generated from NRRL B-16338 (AY650268).S. nassauensis strain LLR-40K-21T cells are non-motile and filamentous, producing a with pale yellow to pale tan substrate mycelium on solid media [S. nassauensis LLR-40K-21T is positive for hydrolysis or degradation of allantoin, casein, esculin, gelatin, hypoxanthine, starch and tyrosine but negative for adenine and xanthine [S. nassauensis strain LLR-40K-21T contains meso-diaminopimelic acid as the diamino acid and N-glycolylmuramic acid as is found in members of the genus Glycomyces. Ribose is the major cell wall sugar. Mannose has also been reported [Glycomyces strains, has not been found in Stackebrandtia [T is dominated by saturated branched chain acids, anteiso-(ai-) C17:0 (26.8%), ai-C15:0 (2.8%), and iso-(i-) C15:0 (8.7%), i-C16:1 (2.1%), i-C16:0 (8.7%), i-C17:0 (9.0%). Unsaturated straight chain acids play only a limited role: C17:1 cis9 (1.8%), and C16:1 cis9 (3.1%). A significant amount of ai-C17:0 2-OH (14.5%) and moderate amounts of hydroxylated fatty acids were also detected. Moderate amounts of saturated components including 10-methyl-branched heptadecanoic acid C16:010 methyl (9.0%) and 10-methyl-C17:0 (1.4%) were also detected. The occurrence of 10-methyl branched heptadecanoic acid and i-branched 1-OH fatty acids is differential for S.nassauensis from members of the genus Glycomyces which lack these acids. Polar lipids identified are phosphatidylglycerol, diphosphatidylglycerol, like in members of the genus Glycomyces, and two additional yet unknown phospholipids are present. Phosphatidylinisitolmanosides (PIM) and phosphatidylglycerol (PI), which are present in the members of the genus Glycomyces, are absent; however, PI is present in S. albiflava [T [et al. [4), MK-10 (H6), MK-11 (H4) and MK-11 (H6), different from the patterns observed from the members of the genus Glycomyces which contain menaquinones with 9-12 isoprene units with various degrees of hydrogenation [The murein of  Genomic  Encyclopedia of  Bacteria and  Archaea  project. The genome project is deposited in the Genomes OnLine Database [This organism was selected for sequencing on the basis of its phylogenetic position, and is part of the Database  and the S. nassauensis strain LLR-40K-21T, DSM 44728, was grown in DSMZ medium 553 (GPHF Medium) [et al. [ Medium)  at 28°C. [et al. .The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website. 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 7,157 overlapping fragments of 1,000 bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the phrap assembler . Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones . Gaps beGenes were identified using Prodigal  as part The genome is 6,841,557 bp long and comprises one circular chromosome with a 68.1% GC content . Of the


Escherichia coli (EHEC) O157:H7 is the causative agent of hemorrhagic colitis and hemolytic uremic syndrome in humans. However, the bacterium can colonize the intestines of ruminants without causing clinical signs. EHEC O157:H7 needs flagella (H7) and hemorrhagic coli pili (HCP) to adhere to epithelial cells. Then the bacterium uses the translocated intimin receptor (Tir) and an outer membrane adhesion (Intimin) protein to colonize hosts. This leads to the attachment and effacement of (A/E) lesions. A tetravalent recombinant vaccine (H7-HCP-Tir-Intimin) composed of immunologically important portions of H7, HCP, Tir and Intimin proteins was constructed and its efficacy was evaluated using a caprine model. The results showed that the recombinant vaccine induced strong humoral and mucosal immune responses and protected the subjects from live challenges with EHEC O157:H7 86-24 stain. After a second immunization, the average IgG titer peaked at 7.2×105. Five days after challenge, E. coli O157:H7 was no longer detectable in the feces of vaccinated goats, but naïve goats shed the bacterium throughout the course of the challenge. Cultures of intestinal tissues showed that vaccination of goats with H7-HCP-Tir-Intimin reduced the amount of intestinal colonization by EHEC O157:H7 effectively. Recombinant H7-HCP-Tir-Intimin protein is an excellent vaccine candidate. Data from the present study warrant further efficacy studies aimed at reducing EHEC O157:H7 load on farms and the contamination of carcasses by this zoonotic pathogen.Enterohemorrhagic  Escherichia coli (EHEC) O157:H7 is a zoonotic enteric pathogen associated with hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in humans. Ruminants are the main reservoir of E. coli O157:H7 which usually colonizes the intestinal tract without causing clinical signs Enterohemorrhagic E. coli O157:H7 E. coli O157:H7 on cattle E. coli O157:H7 E. coli O157:H7 after oral challenge E. coli O157 bacterial ghosts was found to provide protection in a bovine experimental model Reductions in the number of EHECO157:H7 infection in cattle and in feces excreted by asymptomatic shedders can significantly decrease the risk of human exposure to this pathogen E. coli O157:H7colonization of the gastrointestinal epithelium include outer membrane proteins, type III secretion system (T3SS) proteins, flagella, and pili. These proteins are often chosen to construct recombinant vaccines. Among them, intimin (eae gene) and Tir (tir gene) are key colonization factors, which paly significant roles in E. coli O157:H7attachment to host epithelium fliC gene is another interesting virulence factor. It reduces the rate of colonization but not that of overall bacterial shedding E. coli O157:H7 The versatile virulence factors contributing to E. coli O157:H7, and recombinant subunit vaccines consisting of these proteins may hold the key to successful pre-harvest intervention of E. coli O157:H7. To test this hypothesis, a multivalent H7-HCP-Tir-Intimin protein was constructed and expressed for use as a vaccine candidate. A caprine model involving two-month-old goats was established to evaluate the effectiveness of H7-HCP-Tir-Intimin vaccine in the prevention of the colonization and spreading of E. coli O157:H7.Because intimin, Tir, H7 flagellin, and HCP are critical to many of the stages of intestinal colonization by The care of laboratory animals and animal experimentation were performed in compliance with the Jiangsu Administration Guidelines for the Use of Experimental Animals. This study and all procedures were approved by the Animal Ethics Committee of Jiangsu Institute of Veterinary Medicine (SYXK20111101).E. coli O157:H7 86-24 is a well-characterized Shiga-toxin-producing strain. Plasmid Pcold I and pET32 were acquired from TaKaRa Corp. Bacteria are grown in Luria-Bertani (LB) broth and on LB agar (Oxoid) supplemented with 100 µg/mL of ampicillin as needed for selection of recombinant plasmids. E. coli O157:H7 was recovered from a freezer and cultured in brain-heart infusion (BHI) broth.The bacterial strains and plasmids used in this study are listed in fliC, hcpA, tir, and eae were amplified by PCR from DNA isolated from E. coli O157:H7 86-24 strain with primers fliC-P1/fliC-P2, hcpA-P1/hcpA-P2, tir-P1/tir-P2, and eae-P1/eae-P2, respectively. The sizes of the gene fragments were 946 bp, 423 bp, 309 bp, and 811 bp, respectively. Restriction sites for four endonucleases  were incorporated into the PCR primers . Plasmids pET32-fliC, pET32-hcpA, pET32-tir, and pET32-eae were produced in a previous work. The primers and restriction enzymes are described in detail in fliC-hcpA-tir-eae diagram is shown in Fragments of  primers Table 2.E. coli BL21 (Pcold I-fliC-hcpA-tir-eae) was diluted 1∶50 into a flask each containing 200 mL LB broth. When bacteria were grown to the late exponential phase (OD600 = 1.2) at 37°C, the cultures were induced with 0.5 mM IPTG and incubated for additional 12 h at 15°C. The recombinant protein was purified according to the manufacturer’s instructions (Macherey-Nagel Corp.). The bacteria were pelleted by centrifugation at 12,000 g for 5 min and resuspended in 10 ml of 1×Lysis-Equilibration-Wash buffer (LEW) buffer. The concentrated bacterial suspensions were then sonicated  and centrifuged at 12,000 g for 30 min again. The supernatant was collected and purified using His•Bind™ Resin Chromatography according to the manufacturer’s instructions (Macherey-Nagel Corp.). The expected size of recombinant His-H7-HCP-Tir-Intimin was approximately 88 kD. Similarly, recombinant Trx-H7, Trx-HCP, Trx-Tir, and Trx-Intimin proteins were expressed An overnight culture of Specific polyclonal antibodies against His-H7-HCP-Tir-Intimin, Trx-H7, Trx-HCP, Trx-Tir, and Trx-Intimin were produced by subcutaneously injecting 4-month-old New Zealand white rabbits (2 rabbits per protein). Approximately 2 mL of recombinant vaccine containing 1 mg of purified protein was emulsified 1∶1 in Freund complete adjuvant was injected at multiple sites on the animals’ necks. The rabbits received one booster injection with the same amount of antigen emulsified 1∶1 with Freund’s incomplete adjuvant 21 days later. They were bled 10 days after the booster. Sera were filter (0.2 µm) sterilized and stored at −80°C until needed for a Western blotting assay.The purified proteins were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane . The membranes were blocked in 5% nonfat dry milk in Tris-Buffered Saline (TBS), pH 7.2, containing 0.1% Tween 20 (TBST) overnight at 4°C and then incubated with corresponding primary antibodies, including rabbit anti-H7, anti-HCP, anti-Tir and anti-Intimin  for 2 h. After three washes with 0.5% nonfat dry milk in TBST, the membranes were incubated with a 1∶4,000 dilution of HRP-conjugated anti-rabbit IgG . Antigen-antibody complexes were visualized with the 3, 3′-diaminobenzidine (DAB) detection kit . Control immunoblots were performed with pre-immunization rabbit sera.E. coli O157:H7 in their feces. Six goats were injected subcutaneously at multiple sites on their necks with 200 µg/mL recombinant antigen emulsified1∶1(volume/volume) with ISA50V adjuvant  on day 1, and given booster shots 3 weeks later. The remaining six goats were immunized with the same volume of ISA50V adjuvant. Serum samples and fecal samples were collected from all goats twice, on day 1 and again on day 35 (week 5).Twelve 2-month-old male goats were divided into two groups and housed in 4 pens with three goats per pen. Goats were raised in the Animal House of Jiangsu Institute of Veterinary Medicine. On the day before vaccination (day 0), all animals were screened to confirm that they were negative for serum antibodies against His-H7-HCP-Tir-Intimin and that they did not shed 9 CFU E. coli O157:H7 strain 86-24 in 10 mL PBS using a drinking injector. Inoculation of each animal was completed within 2 min. Fecal excretion was monitored daily for two weeks, as described previously E. coli O157:H7. Prior to fecal collection, a large piece of sterile gauze was placed under each cage. Fecal samples  were collected on this sterile gauze in 10 mL PBS, softened by incubation with PBS for 2–4 h at 4°C and then homogenized in a stomacher. The homogenates were centrifuged at 500 g for 15 min to remove fecal materials. The supernatants were centrifuged at 11,000 g for 10 min, and the pellets were resuspended in 1 mL PBS followed by 10-fold serial dilutions. One hundred microliters of 10-fold dilutions were plated onto Sorbitol MacConkey (SMAC) agar plates containing novobiocin, tellurite, and vancomycin E. coli O157 with a latex agglutination test . Duplex PCR of O157:H7 was performed according to the methods developed by our laboratory E. coli O157 was set to 10 CFU/g by enrichment Two weeks after the administration of booster shots, all goats were challenged orally with 10E. coli O157:H7 colonization was evaluated. Twenty-five grams of tissue from the rumen, abomasum, duodenum, jejunum, ileum, cecum, spiral colon, colon, rectum, and rectoanal junction (RAJ) of these experimental goats were collected. Tissue samples were homogenized using a stomacher and 10-fold serial dilutions were prepared. Tissue bacterial load was examined using direct culturing and IMS with O157 Dynabeads as described above in the description in the processing of feces.All the animals were euthanized 14 days after challenge using intravenous administration of 100 mg/Kg pentobarbital sodium. Then Antibody responses specific to H7-HCP-Tir-Intimin were assessed using an enzyme-linked immunosorbent assay (ELISA) as described previously Western blot analysis was used to detect sera from vaccinated and naïve goats to confirm the results of the ELISA. Briefly, H7-HCP-Tir-Intimin protein resolved by PAGE was transferred to nitrocellulose and incubated with sera (1∶500 dilutions) from all experimental goats for 2 h each. Membranes were washed with TBST and incubated with a 1∶8,000 diluted rabbit anti-goat IgG-HRP (KPL Corporation). Immunoblots were visualized using a DAB detection kit .Fecal ELISA was used to detect IgA in feces on day 1 and day 35. ELISA procedures were similar to those described above excepting that fecal samples and rabbit anti-goat IgA-HRP were used instead of goat sera and rabbit anti-goat IgG-HRP. Briefly, samples  were mixed with 5 mL of PBS, vortexed until the pellets were not visible, and then centrifuged. The supernatant was 2-fold serially diluted in PBS and added to ELISA plates (100 µl/well). Rabbit anti-goat IgA-HRP  (AbD Corporation) were used as the secondary antibody. To determine which component of H7-HCP-Tir-Intimin induces IgA secretion, additional ELISA assays were performed. In brief, plates  were coated with Trx-H7, Trx-HCP, Trx-Tir, Trx-Intimin (2.5 µg/mL in coating buffer) overnight at 4°C. The remaining procedures were the same as what were described for fecal IgA ELISA. Differences in immune responses between experimental groups were analyzed using Microsoft Excel, and the t-test was used to analyze antibody responses.2 atmosphere in polystyrene 6-well plates  containing glass coverslips, as previously described 7 bacteria grown overnight in brain-heart infusion (BHI) plus NaHCO3 (BHIN)  at 37°C was added to wells, and the cells were infected for 3 h, washed with PBS to remove unbound bacteria. Fresh medium was added to each well and incubated for additional 3 h and washed with PBS to remove the rest of unbound bacteria. Cells were fixed with 70% methanol/PBS for 5 minutes, and stained with Giemsa for 20 minutes. The coverslips were mounted on glass slides to be observed by light microscopy.For adherence experiment, HEp-2 cells were cultivated at 37°C under 5% COE.coli O157:H7 was incubated with vaccination sera before infection Hep-2 cells. Briefly, E.coli O157:H7 were mixed with 10-fold diluted vaccination sera and incubated at 37°C under 5% CO2 atmosphere for 30 min For experiment of suppressive adherence, the procedures were similar to those described above excepting that fliC-hcpA-tir-eae), indicating the protein was soluble  containing H7-HCP-Tir-Intimin, at 7.2×105  . That goat became negative on day 10.After challenge with  0.0109) Figure 6E. coli O157:H7 carriage in feces ranged from 5.6×104 CFU/g to 3.2×102 CFU/g. After the enrichment and IMS, all intestinal tissues of the placebo-vaccinated animal were positive, but E. coli O157:H7 was not found in any vaccinated animals, indicating that tissue colonization was minimal or undetectable.After euthanasia, direct fecal culture was positive for 5 naive animals. in vitro by HEp-2 cells. For infection experiments, we propagated E.coli O157:H7 overnight in BHIN at 37°C in order to advance adherence. The light microscopy images of infected, Giemsa-stained cells showed obvious impairment of adherence of E.coli O157:H7 after incubation with vaccination sera as compared with naive sera  One report on an E. coli O157 outbreak in Scotland was linked to unpasteurized goat’s milk has been published online (http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=1387). In Greece, E. coli serogroup O157 was used to isolate of 24 strains (1.4%) of which 21 were isolated from bovine milk (2.2%) and 3 from caprine milk (0.7%) E. coliE. coli O157:H7. A vaccine based on the translocon proteins EspA and EspB, and Intimin significantly reduced fecal shedding of E. coli O157:H7 in orally infected sheep E. coli O157:H7 E. coli O157:H7 burden in goats.Because small ruminants are potential reservoirs of E. coli O157:H7 shedding in naturally infected steer The development of multi-component vaccines has become a priority of modern vaccine research. In the current study, a tetravalent recombinant vaccine (H7-HCP-Tir-Intimin) comprising the central region of H7 (H7315), nearly full-length HcpA (HcpA141), the carboxy terminus of Intimin (Intimin270), and the exposed C-end region of Tir (Tir103) was constructed and evaluated. H7 and HCP were chosen because these proteins can block bacterial attachment in the early stage of colonization. Tir is an important T3SS protein. It acts as a receptor for Intimin. Both Tir and Intimin are the preferred choices for recombinant subunit vaccines. In cattle, vaccination with supernatant preparations containing T3SS proteins does decrease E. coli O157 colonization in goats. After the initial inoculation, serum IgG titers increased significantly. In order to improve immune responses, a booster was administered three weeks after primary immunization, the same schedule used for cattle vaccines prepared from the supernatant of bacterial cultures for achieve optimal immunity 5) compared to the titer at the primary immunization (1.4×105). Western blotting was carried out to assess host immune responses, and each serum from all vaccinated goats reacted to H7-HCP-Tir-Intimin, confirming the immunogenicity of the recombinant protein. Experiment of suppressive adherence to HEp-2 cells was used to evaluate the blocking ability of H7-HCP-Tir-Intimin antibody, and each serum from all vaccinated goats reduced the number of adherent bacteria to HEp-2 cells.The data from the current study showed that recombinant H7-HCP-Tir-Intimin can prevent E. coli O157:H7 antigens E. coli in vaccinated goats than in control animals (P = 0.0109) . McNeilly et al. demonstrated that intramuscular vaccination with a combination of intimin-531, the translocon filament protein, EspA, Tir, and H7 flagellin significantly reduced shedding of E. coli O157 from experimentally infected cattle E. coli O157:H7. The vaccine discussed in this paper included four immunogens, but they were expressed separately and then mixed to prepare the vaccine. This way of preparing vaccine is not economically efficient or convenient. A better vaccine design in our study is to prevent both the first adherence of E. coli O157:H7 and the consequent invasion into epithelial cells using animal immunization. For this reason, HCP and H7 were used to block initial adherence and intimin and Tir were used to block invasion and colonization. In order to prepare the vaccine more easily, all individual genes were linked to the same vector of Pcold I to express one protein, in this case H7-HCP-Tir-Intimin. This plasmid vector can be used to express soluble proteins in a manner similar to their natural form . Whether the tetravalent vaccine provides the same efficacy in cattle remains to be proven. Cattle experiments must be designed to confirm its effectiveness. In summary, the tetravalent recombinant H7-HCP-Tir-Intimin vaccine was here found to prevent E. coli O157:H7 intestinal colonization and fecal shedding.Fecal shedding is an important parameter of the effectiveness of recombinant  0.0109) Figure 6E. coli O157:H7, was shown to be involved in bacterial adherence, invasion, hemagglutination, biofilm formation, twitching motility, and extracellular matrix glycoprotein binding E. coli O157:H7 colonization in the mouse gut Present data also show that H7 flagella and HCP play important roles in mitigating bacterial shedding and persistence in the goat intestinal tract. Flagellin has been proposed as a vaccine candidate because of its intrinsic adjuvant activity, which is mediated through TLR5 in vitro and increased shedding from 14-week-old pigs E. coli O157:H7 was cultured in brain-heart infusion (BHI) plus NaHCO3 (BHIN) with gentle shaking. There were more bacteria adhering to HEp-2 under these conditions than in LB with vigorous shaking (data not shown). Collectively, the use of cultural conditions mimicking in vivo environment is essential to the development of a successful animal model for the evaluation of vaccines.It has been reported that culture conditions play an important role in the ability of EHEC O157:H7 to adhere to cultured cells and to epithelial cells in the pig terminal ileum In conclusion, vaccination of goats with recombinant H7-HCP-Tir-Intimin protein reduced fecal shedding of EHEC O157:H7 very effectively. The recombinant protein can be easily produced in large quantities and the production can be standardized for consistent performance. Data from the current investigation warrants further clinical trials aimed at reducing bacterial load on farms and mitigating the contamination of carcasses by EHEC O157:H7.


The disulfide-bonded region (DSR) of HIV-1 gp41 mediates association with gp120 and plays a role in transmission of receptor-induced conformational changes in gp120 to gp41 that activate membrane fusion function. In this study, forced viral evolution of a DSR mutant that sheds gp120 was employed to identify domains within gp120-gp41 that are functionally linked to the glycoprotein association site.AD8 mutant, W596L/K601D, was serially passaged in U87.CD4.CCR5 cells until replication was restored. Whereas the W596L mutation persisted throughout the cultures, a D601H pseudoreversion in the DSR partially restored cell-free virus infectivity and virion gp120-gp41 association, with further improvements to cell-free virus infectivity following a 2nd-site D674E mutation in the membrane-proximal external region (MPER) of gp41. In an independent culture, D601H appeared with a deletion in V4 (Thr-394-Trp-395) and a D674N substitution in the MPER, however this MPER mutation was inhibitory to W596L/K601H cell-free virus infectivity. While cell-free virus infectivity was not fully restored for the revertant genotypes, their cell-to-cell transmission approached the levels observed for WT. Interestingly, the functional boost associated with the addition of D674E to W596L/K601H was not observed for cell-cell fusion where the cell-surface expressed glycoproteins function independently of virion assembly. The W596L/K601H and W596L/K601H/D674E viruses exhibited greater sensitivity to neutralization by the broadly reactive MPER directed monoclonal antibodies, 2F5 and 4E10, indicating that the reverting mutations increase the availability of conserved neutralization epitopes in the MPER.The HIV-1The data indicate for the first time that functional crosstalk between the DSR and MPER operates in the context of assembled virions, with the Leu-596-His-601-Glu-674 combination optimizing viral spread via the cell-to-cell route. Our data also indicate that changes in the gp120-gp41 association site may increase the exposure of conserved MPER neutralization epitopes in virus. The entry of HIV-1 into cells follows receptor binding by the trimeric surface-exposed gp120 glycoprotein, which activates the membrane fusion function of the trimeric transmembrane glycoprotein, gp41. A globular head corresponding to the gp120 trimer encompasses much of the gp41 ectodomain ,4). MembEvidence is accumulating to suggest that the association site formed by the DSR of gp41 and the terminal conserved regions 1 (C1) and 5 (C5) of gp120 -13 act aThe MPER, a highly conserved 23-residue Trp-rich sequence that connects the helical region 2 (HR2) of the gp41 ectodomain to the transmembrane domain, plays an important role in the membrane fusion mechanism . This seAD8-W596L/K601D (WL/KD), by sequential passage in U87.CD4.CCR5 cells. A D601H pseudoreversion in the DSR operated in conjunction with D674E in the MPER to improve gp120-gp41 association and to enable efficient viral spread in culture. The revertant W596L/K601H (WL/KH) and W596L/K61H/D674E (WL/KH/DE) viruses exhibited increased sensitivity to the broadly neutralizing MPER-directed MAbs 2F5 and 4E10. The data indicate that the MPER is functionally linked to the association/activation synapse of gp120-gp41 and suggest that changes in the gp120-gp41 association site may increase the exposure of conserved MPER neutralization epitopes in virus.The aim of this study was to identify determinants that are functionally linked to the “activation synapse”. To this end, we forced the evolution of a DSR mutant virus with disrupted gp120 association, HIV-1AD8 viral replication in U87.CD4.CCR5 cells -independent infection via the endosomal pathway. Twenty-four h after infection, the U87.CD4.CCR5 cells were extensively washed and trypsinized to remove residual adsorbed virus prior to further culture for 10 days. The sequential passaging of the resultant cell-free virus in U87.CD4.CCR5 cells led to restored infectivity after 47 and 30 days in WL/KD cultures 1 and 2 (WLKD-1 and WLKD-2), respectively  prior to the appearance of D674E in the MPER at day 20, which persisted throughout the culture period. The genotypes observed over the 50-day culture period included WL/KH (9/35 clones), W596L/K601H/D674E (WL/KH/DE [10/35 clones]), L85M/W596L/K601H/D674E (LM/WL/KH/DE [6/35 clones]), W596L/K601H/D674G (WL/KH/DG [4/35 clones]), L85M/W596L/K601H (LM/WL/KH [1/35 clones]), and W596L/K601H/D674N (WL/KH/DN [1/35 clones]) , together with W596L and D601H in the DSR, and D674N in the MPER (ΔTW/WL/KH/DN). The ΔTW/WL/KH/DN genotype persisted to day 30 but at days 40 and 50, the dominant genotype was WL/KH/DN (13/18 clones) .The The dominant genotypes were reconstructed in the context of the pAD8 proviral clone. In the case of WLKD-1, cell-free virus-initiated replication in U87.CD4.CCR5 cells was partially restored by D601H in the DSR (WL/KH) and was optimised further by D674E in the MPER (WL/KH/DE) Figure A. The ad10 with respect to WT  to enable detection of subtle changes in fusion function. Consistent with the cell-free virus infectivity data, WL/KD blocked cell-cell fusion, WL/KH exhibited partially restored fusion function and D674N and D674G mutations were inhibitory in a WL/KH context  indicating that gp120-CD4-CCR5 interactions had not been affected by the mutations in gp41  coiled coil in a fusion intermediate conformation of gp41 [P < 0.05, WL/KH and WL/KH/DE versus WT; 2-tailed t test, unequal variances). Notably, WL/KH and WL/KH/DE exhibited markedly greater sensitivity to neutralization by the MPER-specific brNAbs 2F5 and 4E10 when compared to the WT. These data are consistent with structural changes in the gp120-gp41 complex that increase the availability of neutralization targets in the MPER of gp41.To determine whether WL/KH and WL/KH/DE are associated with structural changes in gp41, neutralizing agents were used to probe functional virion-associated gp120-gp41 complexes. Virus stocks, produced by transfecting 293T cells with pAD8 infectious clones, were adjusted to produce ~ 1.5 × 101 Figure . Small,  of gp41 ,44, wereThe forced evolution of WL/KD mutant viruses with severely disrupted gp120-gp41 association led to the emergence of replication-competent revertants containing a D601H pseudoreversion in the DSR plus D674E or D674N 2nd site mutations in the MPER. In a second culture, revertant clones contained the ΔT394-W395 deletion in V4 in addition to D601H and D674N. The WL/KH and WL/KH/DE viruses exhibited greater sensitivity to the brNAbs, 2F5 and 4E10, indicating that the restoration of function was associated with structural changes in Env that increase the accessibility of neutralization epitopes within the MPER. Our data reveal a functional linkage between the DSR and MPER of gp41 and point to a novel approach for improving the accessibility of conserved neutralization epitopes within the MPER in a virion context.The severe shedding phenotype of WL/KD is likely to have resulted from the combined effects of decreased hydrophobic sidechain bulk and an additional negative charge in the contact site, that destabilizes gp120-gp41 association. Phenotypic analysis of the revertant genotypes indicated that D601H is a key evolutionary step that partially restores gp120-gp41 association. Histidine at 601 introduces an imidazole moiety into the association site, which would partially compensate for the loss of the indole ring of Trp-596 and removes the negative charge contributed by Asp-601. Interestingly, Leu-596 was maintained in both long-term cultures, indicating that the smaller hydrophobic sidechain is preferred at this position when His is present at 601. While His is expected to be neutral at physiological pH, its imidazole ring may become protonated if it is proximal to acidic residues. In this case, the D601H mutation would also restore positive charge to position 601, which may also lend stability to the gp120-gp41 association site. The combination of His-601 with D674E led to improved single-cycle and multi-cycle cell-free virus-initiated infectivity without detectable further improvement in gp120-gp41 association. The MPER mutation therefore appears to act at the level of virus entry. Highly mutable viruses, such as HIV-1 have the capacity to overcome fitness defects via multiple evolutionary pathways. It was therefore notable that we found a functional link between positions 601 and 674 of gp41 in parallel cultures, indicating a reproducible genetic link between the DSR and MPER. However, we cannot rule out the possibility that additional solutions to restore the gp120-gp41 interaction would be revealed with more independent evolution cultures of WL/KD.Nuclear magnetic resonance studies of synthetic MPER peptides suggest that in membranes, the MPER is a metastable L-shaped structure comprised of N- and C-terminal helical segments that are connected via a hinge composed of Phe-673, which is buried in the lipid phase, and a polar residue at position 674, which is solvent-exposed Figure  PDB entA, thereb10) but significant increase in C34 sensitivity was observed for WL/KH and WL/KH/DE viruses. Subtle alterations to the function of HR1 during fusion, such as prehairpin coiled coil formation, may accompany the WL/KH and WL/KH/DE changes.Mutational studies have indicated that the DSR is associated with the C1 and C5 regions of gp120 -13,35, wenv genotypes coexist with WL/KH in the WLKD-2 culture even though the former exhibited lower cell-free virus infectivity. It is interesting that the boosts to infectivity associated with the addition of D674E to W596L/K601H did not correlate with enhanced cell-cell fusion activity, where the cell-surface expressed glycoproteins function independently of other virion components. These data are consistent with a Leu-596-His-601-Glu-674 functional interaction that is dependent on virion assembly and the structural constraints imposed on the Env ectodomain, including the MPER, by Gag-gp41 cytoplasmic tail interactions [In contrast to the data obtained with cell-free virions, replication levels at  or better (WL/KH/DE) than those of WT were achieved by the revertants when infections were initiated with VSV G pseudotyped viruses. In this latter system, the initial infection rounds will be largely mediated by the highly fusogenic VSV G present in the viral envelope thereby normalizing WT and mutant virus infectivity, while subsequent infection rounds will be mediated by gp120-gp41 present on virions transmitted directly from cell to cell via virological synapses in addition to nascent cell-free virions. Direct cell-cell viral transmission has been calculated to be at least 8-times more efficient than cell-free viral spread and is believed to be due to higher effective multiplicity of infection and virus viability within virological synapses . It may ractions -42.i) direct interaction with the MPER in neutralization-sensitive viral strains; ii) Env-receptor interaction-triggered MPER accessibility to brNAb in resistant strains [The WL/KH (and WL/KH/DE) mutations were associated with increased sensitivity to the 2F5 and 4E10 MPER-directed brNAbs, indicating a structural change in Env that increases MPER accessibility. Two neutralization mechanisms have been proposed for 2F5 and 4E10:  strains . We have strains .MPER-specific brNAbs are of particular interest to the HIV-1 vaccine field due to the conserved nature of their epitopes and their neutralization breadth ,61-64. BWe previously reported that the contributions of Trp-596 and Lys-601 to gp120-gp41 association and membrane fusion are influenced by sequence changes in V1, V2, and V3 , which aWe have found, by using a forced virus evolution approach, evidence for functional crosstalk between the gp120-gp41 association site and the MPER. Our data indicate that looser gp120-gp41 association due to the WL/KH mutation within the DSR is linked to enhanced neutralization by brNAbs directed to the MPER. Our work points to a new approach for improving the accessibility of conserved MPER neutralization epitopes in virion-based immunogens.AD8 Env expression vector, pCDNA3.1-AD8env, is described elsewhere [env was derived by religation of the end-filled HindIII and EcoRI sites of pCDNA3.1-AD8env. Mutants of the pAD8 infectious clone (obtained from K. Peden [EcoRI-BspMI env-containing fragment from pCDNA3.1-AD8env vectors into pAD8. In vitro mutagenesis of the gp41 region was carried out using the Quikchange protocol (Stratagene).The CMV promoter-driven HIV-1lsewhere . pΔKAD8eK. Peden ) were pr2 culture flasks. The supernatants were assayed for RT activity [2 culture flasks were inoculated with the HIV-VSV G pseudotypes, and then, at 24-h postinfection, trypsinized to remove surface-adsorbed virions. The cells were replated and then cultured for 10 days. The culture supernatants were assayed for RT activity or p24 content at various time points. For long-term cultures of viral mutants, the day-10 cell-free culture supernatants were filtered (0.45 μm pore size) and normalized according to RT activity prior to the next passage . Genomic DNA was extracted from infected cells using Qiagen DNeasy Blood and Tissue kit. The viral DNA fragment encompassed by nucleotides 5954–9096 (HIV-1HXB2R numbering convention) was PCR-amplified using Expand HiFi (Roche) and the primers, 5’-GGCTTAGGCATCTCCTATGGCAGGAAGAA (Env1A) and 5’-TAGCCCTTCCAGTCCCCCCTTTTCTTTTA (Env1M) [env (KpnI-XbaI) and the entire env open reading frame sequenced using ABI BigDye terminator v3.1.Virus stocks were prepared by transfecting 293T cell monolayers with pAD8 infectious clones using Fugene 6 or Fugene HD (Roche). Virus-containing transfection supernatants were normalized according to RT activity, and then used to infect U87.CD4.CCR5 astroglioma cells , using env vectors, 293T cells were lysed for 10 min on ice in PBS containing 1% Triton X-100, 0.02% sodium azide, 1 mM EDTA. The lysates were clarified by centrifugation for 10 min at 10,000 ×g at 4°C prior to SDS-PAGE under reducing conditions. The proteins were transferred to nitrocellulose and blotted with antibodies C8 to gp41 [Twenty four h after transfection with pΔKAD8 to gp41  and DV-0 to gp41  , and then labeled for 45 min with 150 μCi Tran-35S-label . The cells were washed and then chased in complete medium for 5 h prior to lysis. Cell lysates and clarified culture supernatants were immunoprecipitated with pooled IgG from HIV-1-infected persons and protein G Sepharose and subjected to SDS-PAGE in the presence of β-mercaptoethanol. The labeled proteins were visualized by scanning in a Fuji phosphorimager.293T cells were transfected with pΔKAD8env and the bacteriophage T7 RNA polymerase expression vector, pCAG-T7 [luc, a bicistronic vector that expresses human CD4 from a CMV promoter and firefly luciferase from a T7 promoter [Cell-cell fusion assays were conducted as previously described . Briefly pCAG-T7 . BHK21 t pCAG-T7 ) and pT4promoter . At 24 h6 relative light units (RLU) following infection of TZM-bl cells, were mixed with an equal volume of serially diluted IgG or C34 peptide and incubated for 1 h at 37°C. One hundred μl of the virus-IgG mixtures was then added to TZM-bl cells (104 cells in 100 μl per well of a 96-well tissue culture plate) and incubated for 2 days prior to lysis and assay for luciferase activity . For experiments with the CCR5 antagonist maraviroc (NIH AIDS Research and Reference Reagent Program [Purified IgG of brNAbs 2F5 , 4E10 6 and IgGbbrNAb: Broadly neutralizing monoclonal antibody; C: Conserved region of gp120; DSR: Disulfide bonded region; Env: HIV-1 envelope glycoprotein; HR1: HR2, Helical regions 1 and 2 of gp41, respectively; mAb: Monoclonal antibody; MPER: Membrane-proximal ectodomain region; RT: Reverse transcriptase; V: Variable region of gp120; VSV G: Vesicular stomatitis virus glycoprotein G.The authors declare that they have no competing interests.AIK, HD and PP conceived the study. AIK, AKB-M, HD and PP wrote the manuscript. AIK, AL, DNH, AKBM, HD and PP acquired experimental data and performed the data analysis. All authors read and approved the final manuscript.


Proteobacteria. Several T6SSs are tightly regulated by various regulatory systems at multiple levels. However, the signals and/or regulatory mechanisms of many T6SSs remain unexplored. Here, we report on an acid-induced regulatory mechanism activating T6SS in Agrobacterium tumefaciens, a plant pathogenic bacterium causing crown gall disease in a wide range of plants. We monitored the secretion of the T6SS hallmark protein hemolysin-coregulated protein (Hcp) from A. tumefaciens and found that acidity is a T6SS-inducible signal. Expression analysis of the T6SS gene cluster comprising the imp and hcp operons revealed that imp expression and Hcp secretion are barely detected in A. tumefaciens grown in neutral minimal medium but are highly induced with acidic medium. Loss- and gain-of-function analysis revealed that the A. tumefaciens T6SS is positively regulated by a chvG/chvI two-component system and negatively regulated by exoR. Further epistasis analysis revealed that exoR functions upstream of the chvG sensor kinase in regulating T6SS. ChvG protein levels are greatly increased in the exoR deletion mutant and the periplasmic form of overexpressed ExoR is rapidly degraded under acidic conditions. Importantly, ExoR represses ChvG by direct physical interaction, but disruption of the physical interaction allows ChvG to activate T6SS. The phospho-mimic but not wild-type ChvI response regulator can bind to the T6SS promoter region in vitro and activate T6SS with growth in neutral minimal medium. We present the first evidence of T6SS activation by an ExoR-ChvG/ChvI cascade and propose that acidity triggers ExoR degradation, thereby derepressing ChvG/ChvI to activate T6SS in A. tumefaciens.The type VI secretion system (T6SS) is a widespread, versatile protein secretion system in pathogenic  Agrobacterium tumefaciens. Our data suggested that ExoR represses ChvG sensor kinase by physical interaction and the acid-induced degradation of periplasmic ExoR may derepress ChvG to activate T6SS by phosphorylation of the ChvI response regulator. The activation of T6SS by an acidic signal present in the wound site and intercellular space of plants implicates a role of T6SS during Agrobacterium–plant interactions. In view of the conservation of ExoR and ChvG/ChvI and wide distribution of T6SS in α-Proteobacteria, including many animal and plant pathogens and symbionts, the regulation of T6SS by the ExoR-ChvG/ChvI cascade may be a universal regulatory mechanism in these bacteria.The bacterial type VI secretion system (T6SS) has diverse functions that contribute to the survival or fitness of many pathogenic bacteria in response to environmental cues. Numerous studies have shown that T6SS is highly regulated via multiple mechanisms, but the regulatory mechanisms of most T6SSs remain unknown. In this study, we discovered that T6SS is activated by acidity via an ExoR-ChvG/ChvI cascade in a plant pathogenic bacterium,  ProteobacteriaPathogenic bacteria have evolved several specialized secretion systems to transport protein or DNA across membranes to extracellular milieu or even to the host cells in response to specific environmental cues. Among the 6 types of secretion systems, named type I to VI (T1SS to T6SS) identified in Gram(−) bacteria Escherichia coli, the sci1 T6SS gene cluster is under the control of an epigenetic switch regulated by iron availability through Fur- and Dam-dependent methylation Burkholderia mallei by VirA/VirG, Edwarsiella tarda by EsrA/EsrB, and Salmonella enterica by SsrA/SsrB Pseudomonas aeruginosa HSI-1 T6SS P. aeruginosa, HSI-1 T6SS is posttranslationally regulated by serine/threonine kinase PpkA and the cognate phosphatase PppA via threonine phosphorylation on a forkhead-associated protein, Fha1 The diverse functions of T6SS are reflected by its regulation by multiple mechanisms. T6SS is regulated at epigenetic, transcriptional, posttranscriptional, and posttranslational levels Agrobaterium tumefaciens is a soil bacterium causing crown gall disease in a wide range of plants. It integrates transferred DNA (T-DNA) from the tumor-inducing plasmid into the host genome A. tumefaciens encounters signals such as acidity, monosaccharides, and phenolic compounds released from plant wound sites, the VirA/VirG two-component system, in cooperation with periplasmic sugar binding protein ChvE, is activated to induce the expression of virulence (vir) genes to direct the transfer of T-DNA into host cells vir genes encoded by tumor-inducing plasmid, the chromosome-encoded ChvG/ChvI two-component system is responsible for the expression of acidity-inducible genes, including aopB, encoding an outer membrane protein; pckA, encoding phosphoenolpyruvate carboxykinase; and virGα-ProteobacteriaSinorhizobium meliloti, the ChvG ortholog ExoS is the sensor kinase and with the response regulator ChvI, functions as the positive regulator for synthesis of exopolysaccharide succinoglycan, promoting biofilm formation and motility S. meliloti, ExoR regulates its own expression through ExoS/ChvI A. tumefaciensThe ChvG/ChvI two-component system is highly conserved in A. tumefaciens remains largely unknown. Previously, we found the secretion of the T6SS hallmark protein hemolysin-coregulated protein (Hcp) in A. tumefaciens grown under various conditions, including nutrient-rich or minimal medium at low (19°C) or room temperature imp operon is higher under acidic than neutral minimal medium conditions imp gene expression is responsible for activation of T6SS secretion and the regulatory mechanisms underlying T6SS expression and activity are unknown.How T6SS is regulated in A. tumefaciens T6SS is regulated by plant-derived signals and if so, the underlying regulatory mechanism. T6SS-mediated Hcp secretion was almost silent with A. tumefaciens grown in neutral minimal medium but was induced by acidity. Further molecular analysis revealed that T6SS is activated by the ChvG/ChvI two-component system, with the sensor kinase ChvG negatively regulated by the periplasmic repressor ExoR. Importantly, we provide the first evidence that acidity induces ExoR degradation, which then may derepress ChvG to activate T6SS through a ChvI response regulator in a phosphorylation-dependent manner. This activation of T6SS by an acidic signal present in plant wound sites and apoplasts (intercellular space) suggests its potential role during Agrobacterium infection or replication near or inside plants.In this study, we aimed to investigate whether the expression and secretion of A. tumefaciens grown in acidic minimal medium  A. tumefaciens by Hcp secretion assay A. tumefaciens can sense plant-derived signals, including acidity, monosaccharides, and phenolic compounds, which are critical components in the acidic minimal medium  for vir gene expression, we first tested whether any of these 3 signals plays a role in regulating T6SS activity. A. tumefaciens wild-type strain C58 cells cultured overnight in AB-MES (pH 7.0) was sub-cultured in neutral (pH 7.0) or acidic (pH 5.5) AB-MES minimal medium with or without monosaccharides or acetosyringone (AS) phenolics for 6 h. Secretion of Hcp was abundant from A. tumefaciens cells grown in acidic minimal medium (pH 5.5)  , with ba(pH 5.5) . Intriguth no AS . The nonth no AS . TherefoEdwardsiella tarda and Vibrio cholera, along with other studies, revealed about a dozen conserved components essential for mediating T6SS secretion A. tumefaciens, the T6SS gene cluster comprises 2 divergently transcribed operons: imp, encoding 14 genes (atu4343 to atu4330); and hcp, encoding 9 genes (atu4344 to atu4352)  and 3 by the hcp operon  with acidity   were barely detected when grown in neutral minimal medium, whereas those encoded by the hcp operon  were expressed at substantial levels under this growth condition  . To exam pH 5.5) . As contondition . The levl medium . TherefoA. tumefaciens. The ChvG/ChvI two-component system functions to regulate certain acidity-inducible genes, including the system itself, and may serve as a global pH sensor in A. tumefaciensΔchvG and ΔchvI in-frame deletion mutants for the two-component system and examined the effects on T6SS expression and Hcp secretion. Because ΔchvG and ΔchvI are sensitive to a nutrient-rich or acidic environment imp or hcp operon showed different basal levels in neutral minimal medium , and their protein levels were all further reduced with chvG or chvI deletion  /ChvI signaling is negatively regulated by ExoR in  operons , which i operons , which i pH 7.0) . Therefoimp and hcp operons. We used electrophoretic mobility shift assay (EMSA) and incubated the purified His-ChvI recombinant protein with a 230-bp DNA fragment derived from the intergenic region of the imp and hcp operons but detected no binding activity  in A. tumefaciens wild-type C58 to determine whether this phospho-mimic ChvI was sufficient to trigger T6SS expression and secretion in neutral medium, the secretion-repression condition. In parallel, we overexpressed the sensor kinase ChvG and wild-type ChvI in C58 as controls. Both the mRNA and protein levels of the analyzed imp genes were significantly induced with ChvG and ChvI(D52E) overexpression as compared with vector expression alone , with inactivation of the ChvI phosphorylation site, further reduced imp gene expression at both mRNA and protein levels.To determine whether the binding of phospho-mimic ChvI(D52E) to the T6SS promoter region on alone . In conthcp operon was regulated differently from the imp operon with the overexpression strains. As expected, hcp operon expression was increased at both mRNA and protein levels with ChvG and ChvI(D52E) overexpression  and phospho-inactive ChvI(D52A) were insensitive to the acidity in regulating T6SS, with evidence that ChvI(D52E) was constitutively active and ChvI(D52A) defective in T6SS expression and secretion in both neutral and acidic medium  at the C terminus was driven by a constitutively active lac promoter from pRL622 (Plac-ChvG-HA), and ExoR was expressed by an isopropyl-beta-D-thiogalactoside (IPTG)-inducible trc promoter from pTrc200 (Ptrc-ExoR). The ectopic expression of ExoR and ChvG also allowed us to monitor their protein expression and stability at posttranscriptional levels. The AB-MES (pH 7.0) overnight-grown bacterial culture was subcultured in neutral (pH 7.0) or acidic (pH 5.5) AB-MES medium for 3, 6, 12, and 24 h and collected for western blot analysis. Both neutral and acidic medium maintained the pH over this time because we detected only a slight decrease of pH after 12- to 24-h growth , and this interaction is critical for inhibiting ExoS/ChvI signaling A. tumefaciens grown under neutral conditions. When the acidic signal is sensed, the degradation of ExoRm may thereby allow the inner-membrane–associated ChvG sensor kinase to activate the ChvI response regulator and induce T6SS expression and secretion activity.The inverse association of ExoR and ChvG protein level, together with the epistasis of A. tumefaciens and S. meliloti and identified the putative amino acid residues in A. tumefaciens ExoR that may be critical for interaction with ChvG (exoR mutants encoding ExoR variants with amino acid substitution mutations at 2 specific residues in the Sel1 repeat of ExoR (G73 and S153) that are responsible for interaction with ExoS (ChvG) and result in increased ExoS(ChvG)/ChvI activity in S. melilotiΔchvG ΔexoR mutant to determine whether these amino acid residues are critical for ChvG protein levels in neutral minimal medium. ChvG-HA protein level was lower with complementation of pExoR expressing wild-type ExoR than with the vector control  and ExoR(S153Y) variants than with wild-type ExoR  revealed an inverse association of T6SS and VirB/D4 T4SS that is highly induced by AS T6SS is involved in diverse functions, including promoting or repressing virulence, forming biofilm, and inducing cytotoxicity in eukaryotic or prokaryotic hosts. Many T6SSs of pathogenic bacteria are induced by host signals, which suggests their functions during bacteria–host interactions A. tumefaciens. We also noted that T6SS secretion activity depends on growth phase, with Hcp secretion greatly reduced during the late logarithmic phase . Thus, T6SS is regulated by multiple factors via a complex regulatory network in a free-living environment or during Agrobacterium–plant interactions.Our previous study did not reveal the suppression of Hcp secretion at pH 7.0 A. tumefaciens. This discovery adds T6SS genes to the list of ChvG/ChvI-regulatory genes in A. tumefaciens. ExoR, ChvG/ChvI, and T6SS are widely distributed in α-Proteobacteria, which includes several animal and plant pathogens, as well as plant symbionts By loss- and gain-of-function studies, we demonstrated that T6SS is regulated positively by the ChvG/ChvI two-component system and negatively by ExoR in imp genes found in previous microarray analyses hcp operon-encoded genes as acidity-regulated genes. By investigating both the mRNA and protein levels in response to pH change together with loss- and gain-of-function studies, our data strongly argue that acid-induced imp operon expression is regulated at mRNA levels via transcriptional activation by the ChvG/ChvI two-component system. However, the hcp operon is regulated by a ChvG/ChvI pathway and by an unknown mechanism responsible for its basal expression even in the secretion-repression condition . Thus, acid-induced T6SS secretion is mainly controlled by the expression of imp-encoding proteins constituting the T6S machinery. In addition, the higher induction of mRNA than protein levels by acidity , or phospho-inactive (D52A) variants of ChvI could increase both mRNA and protein levels of hcp operon genes, which suggests that the phosphorylated state of ChvI is not required for upregulation of hcp operon expression. Because wild-type ChvI without detectable binding activity to the T6SS promoter region can enhance the level of proteins encoded by the hcp operon in neutral medium, where ChvG is repressed by ExoR, ChvI may not directly regulate the expression of the hcp operon. In view of the chvG/chvI-independent basal level expression of hcp-operon proteins, both phosphorylated and non-phosphorylated ChvI may positively influence the expression by interacting with a yet-to-be identified regulator(s) of hcp operon. Future investigation should aim to identify the cis-elements critical for ChvI binding and elucidate the molecular details of how ChvI coordinates the regulation of the expression of the 2 operons from the shared or overlapped regulatory region.More strikingly, the phosphorylated state of ChvI was required for inducing p operon . For theα-Proteobacteria, such as Brucella, Bartonella, Sinorhizobium, and Rhizobium, which suggests a conserved negative regulation of ChvG/ChvI by ExoR among these bacteria S. meliloti, exoR is involved in sensing ammonia or calcium signals for derepressing the expression of ExoS/ChvI target genes lpsS and exoBartonella henselae BatR/BatS at the physiological pH of blood (pH 7.4) Brucella abortus sensor kinase BvrS at the late logarithmic phase A. tumefaciens ChvG/ChvI in response to the acidic signal S. melilotiA. tumefaciens in this study.Orthologs of ExoR and the ChvG/ChvI two-component system are present in many chvG to exoR in regulating T6SS expression and secretion indicates that exoR functions upstream of chvG in sensing acidity to regulate T6SS. The disruption of A. tumefaciens ExoR amino acid residues critical for interacting with ChvG causes the increased ChvG protein levels and T6SS expression/secretion, which suggests that ExoR negatively regulates ChvG/ChvI signaling by direct binding to ChvG. This conclusion is consistent with the finding that the physical interaction of ExoR–ExoS(ChvG) is important in repressing ExoS/ChvI activity in S. melilotiexoR, ExoR may negatively regulate ChvG protein levels by directing binding.The epistasis of lac promoter  activity exoR or loss of binding to ExoR variants when grown in neutral medium must be regulated by mRNA stability and/or translational efficiency of chvG, but the potential regulations await future investigation.Because endogenous ExoR is not detectable by antibody against omosomes , ExoR prc medium  [36], thA. tumefaciens senses the acidic signal, ExoR loses its activity in repressing ChvG, thereby allowing the ChvG sensor kinase to relay ChvI signaling for T6SS activation. Periplasmic ExoRm may be more stable in neutral pH and rapidly degraded when sensing acidity. However, whether acid-triggered ExoRm degradation is the key or responsible for activating ChvG/ChvI signaling is not yet verified. We do not exclude that acid may also induce a conformational change of ExoR or even ChvG to regulate ChvG/ChvI signaling. Thus, it remains important to visualize the endogenous expression of ExoR and ChvG in response pH changes by pulse-chase analysis To this end, we propose that acid-induced ExoRm degradation is responsible or one factor for the increased ChvG protein levels or activity and T6SS activation. As illustrated in our proposed model , in the A. tumefaciens strains were grown in 523 medium ΔchvG, ΔchvI, and ΔchvG ΔexoR mutant strains were grown in AB-MES minimal medium, which contains 3 g K2HPO4, 1 g NaH2PO4, 1 g NH4Cl, 0.15 g KCl, 0.01 g CaCl2, 0.3 g MgSO4-7H2O, 2.5 mg FeSO4-7H2O, 9.76 g 2-(N-morpholine)ethanesulfonic acid, and 20 g glucose per liter (pH 7.0) A. tumefaciens cells grown overnight in AB-MES medium (pH 7.0) with appropriate antibiotics were harvested by centrifugation  and resuspended in AB-MES medium (pH 7.0 or 5.5) without any antibiotics at ∼0.1 optical density 600 nm (OD600). After growth for 6 h at 25°C, cells were harvested, and proteins secreted into the culture medium were precipitated with trichloroacetic acid (TCA) as described A. tumefaciens cells were resuspended in AB-MES medium (pH 7.0 or 5.5) containing chloramphenicol (100 µg/ml) at OD600 ∼0.5 from A. tumefaciens cells grown overnight in AB-MES medium (pH 7.0) with appropriate antibiotics. Cells were harvested after growth for 1, 2, and 3 h at 25°C. For A. tumefaciens, the concentration of the antibiotic gentamycin was 50 µg/ml; spectinomycin, 250 µg/ml; and chloramphenicol, 100 µg/ml, and for Escherichia coli, the concentration for gentamycin was 50 µg/ml; spectinomycin, 100 µg/ml; and kanamycin, 20 µg/ml.Plasmids, bacterial strains and primers are in supplemental chvG, pJQ200KS-ΔchvI, and pJQ200KS-ΔexoR were created by ligating the XbaI/BamHI-digested PCR product 1 (∼500 bp DNA fragments upstream of each target gene) and BamHI/XmaI-digested PCR product 2 (∼500 bp DNA fragments downstream of each target gene) into XbaI/XmaI sites of pJQ200KS The plasmids pJQ200KS-ΔchvG and chvI genes containing the ribosomal-binding sequence (RBS) and open reading frame (ORF) were digested by XhoI/XbaI and cloned into the same sites of pRL662 chvG DNA fragment containing the RBS and ORF (without a stop codon) was PCR-amplified, digested by SacI/XbaI, and cloned into the same sites of pBluescript SK(+)-HA. The resulting plasmids were further digested by XhoI/HindIII and cloned into the same sites of pRL662 to create the plasmid pChvG-HA.The PCR products of exoR containing its RBS and ORF were digested by XmaI/XbaI and cloned into the same sites of pTrc200 E. coli BL21(DE3) fha1, atu4343, atu4349, and rpoA was PCR-amplified and cloned into the same sites of pET22b(+) with appropriate enzyme sites. The clpV ORF (without a stop codon) was PCR-amplified, digested by HindIII, and cloned into pET22b(+), which was first digested by NdeI, followed by Klenow repair, and finally digested by HindIII. N-terminal His-tagged wild-type and D52E ChvI were constructed by PCR amplification of the full-length wild-type or D52E ChvI with flanking NdeI/XhoI restriction sites and cloning into pET28a (Novagen).The PCR products of exoR ORFs were PCR-amplified (by primers AD ExoR F & AD ExoR R), digested (NdeI/XhoI), and cloned into the NdeI/XhoI sites of pGADT7 for N-terminal fusion to the activation domain (AD), pGADT7-ExoR, pGADT7-ExoRG73C, pGADT7-ExoRS153Y and pGADT7-ExoRG73C/S153Y. For plasmid pGBKT7-ChvG-peri, the DNA fragment encoding the periplasmic domain of ChvG (71–278 a.a.) was PCR-amplified with primers BD ChvGperi F & BD ChvGperi R, digested with NdeI/BamHI, and cloned into the same sites of pGBKT7.For the constructs used for yeast two-hybrid, various A. tumefaciens strains grown in AB-MES minimal medium (pH 7.0 or pH 5.5) was extracted by the hot-phenol method ΔΔCt− method Total RNA from A. tumefaciens C58 via double crossover with the suicide plasmid pJQ200KS All in-frame deletion mutants were generated in E. coli BL21(DE3) followed by purification with an Ni2+-NTA column (Novagen) as described The detailed methods of plasmid construction, overexpression, and purification of His-tagged Fha1, Atu4343, ClpV, Atu4349, and RpoA proteins for antibody production will be published elsewhere . In brief, the expression vector pET22b(+) was used to overexpress His-tagged proteins driven by the T7 promoter with IPTG induction in E. coli BL21(DE3) cells for protein expression. Briefly, cultures were induced with 0.4 mM IPTG for 3 h at 37°C, and cells were lysed by use of the French Press  as described The pET28a-ChvI and pET28a-ChvI(D52E) plasmids were transformed into A. tumefaciens; ExoR S. meliloti; or monoclonal antibody against HA (Sigma). The secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit or anti-rabbit IgG (Chemicon), and signals were detected by use of the Western Lightning System . Chemiluminescent bands were visualized by use of X-ray film  or the BioSpectrum AC Imaging System  to detect and quantify the photon intensity of protein signals.Proteins were resolved by 13% glycine-SDS-PAGE and western blot analysis was performed as described imp and hcp operons and the control DNA fragment were PCR-amplified with the primers T6SSF/T6SSR and Atu4353F/Atu4353R, respectively, and purified by use of the PCR DNA fragment extraction kit . The T6SS regulatory region and control DNA fragments were digested with XhoI and BamHI, respectively, and filled in with [α-32P]dCTP and unlabeled dTTP, dATG, and dGTG with the klenow fragment of DNA polymerase I. The labeled 32P-labeled DNA fragments were purified by use of G50 Mini columns (Geneaid). Labeled DNA fragments (1.5 ng) were incubated with purified ChvI protein (11 to 150 ng) in 10 µl binding buffer  for 20 min at room temperature and then analyzed on 5% Tris-borate-EDTA non-denaturing acrylamide gels at 4°C. The separated DNA–protein complex was dried by use of a gel dryer and visualized by use of X-ray film (Kodak).The 230-bp intergenic region of the chvI(D52E) and chvI(D52A) DNA fragments were digested by NdeI/XbaI and cloned into the same sites of pRL662 to create the plasmids pChvI(D52E) and pChvI(D52A). The exoR(G73C), exoR(S153Y), and exoR(G73C S153Y) DNA fragments were digested by XmaI/XbaI and cloned into the same sites of pRL662 to create the plasmids pExoR(G73C), pExoR(S153Y), and pExoR(G73C S153Y).The DNA fragment encoding ChvI(D52E), ChvI(D52A), ExoR(G73C), or ExoR(S153Y) mutations was created by PCR-based site-directed mutagenesis as described A. tumefaciens cellular fractions was as described Isolation of Saccharomyces cerevisiae strain AH109. The transformants were selected by their growth on synthetic dextrose (SD) minimal medium lacking tryptophan (Trp) and leucine (Leu) (SD-WL medium). The positive interaction of expressed fusion proteins was determined by their growth on SD lacking Trp, Leu, adenine (Ade), and histidine (His) (SD-WLHA medium) at 30°C for at least 2 days.The Matchmaker yeast two-hybrid system was used according to the user manual instructions . Each of the plasmid pairs were co-transformed into hcp (1136219), rpoA (1133961), virE2 (1137513), icmF (1136206), fha1 (1136209), atu4343 (1136217), clpV (1136218), atu4349 (1136223), chvG (1132071), chvI (1132072), exoR (1133753), chvH (1134591)Genebank accession numbers for genes: Genebank accession numbers for proteins: Hcp (NP_356310), RpoA (NP_354899), VirE2 (NP_396510), IcmF (NP_356323), Fha1 (NP_356320), Atu4343 (NP_356312), ClpV (NP_356311), Atu4349 (NP_356306), ChvG (NP_353072), ChvI (NP_353073), ExoR (NP_354703), ChvH (NP_355493)Figure S1The effects of acetosyringone (AS) and different carbon sources on T6SS expression and Hcp secretion. (A) Total proteins isolated from Agrobacterium tumefaciens wild-type C58 grown in AB-MES (pH 7.0 or pH 5.5) supplemented with indicated carbon sources at 25°C for 6 h were resolved by glycine-SDS-PAGE, followed by western blot analysis with antibodies against C-IcmF, Fha1 (filled arrow), Atu4343, ClpV, Hcp, Atu4349, and RpoA. (B) Total and secreted (Sup) proteins isolated from C58 grown in AB-MES (pH 7.0 or pH 5.5) with glycerol, DMSO (used to dissolve acetosyringone [AS]), or 200 µM AS at 25°C for 6 h were resolved by glycine-SDS-PAGE, followed by western blot analysis with antibodies against C-IcmF, Fha1 (filled arrow), Atu4343, ClpV, Hcp, Atu4349, VirE2, and RpoA. RpoA was used as an internal control. The positions of molecular mass markers (in kDa) are indicated on the left.(TIF)Click here for additional data file.Figure S2T6SS expression and Hcp secretion are concomitantly upregulated by acidity in A. tumefaciens Ach5 and 1D1609 strains. (A) Secreted (Sup) proteins isolated from A. tumefaciens wild-type C58, Ach5, 1D1609 strains grown in AB-MES (pH 7.0 or pH 5.5) at 25°C for 6 h were resolved by glycine-SDS-PAGE, followed by western blot analysis with antibodies against Hcp and RpoA. (B) Total proteins isolated from the strains and conditions described in (A) were resolved by glycine-SDS-PAGE, followed by western blot analysis with antibodies against C-IcmF, Fha1 (filled arrow), Atu4343, ClpV, Hcp, and RpoA. RpoA is a non-secreted protein used as an internal control. The positions of molecular mass markers (in kDa) are indicated on the left.(TIF)Click here for additional data file.Figure S3EMSA of the binding specificity of ChvI and T6SS regulatory region. The 32P-labeled 230-bp intergenic region between the imp and hcp operons or DNA fragment derived from the atu4353 coding region was incubated without or with increasing levels of purified recombinant variant ChvI(D52E) protein (50 and 150 ng). The shifted band is indicated by *.(TIF)Click here for additional data file.Figure S4Quantification of levels of hcp operon-encoded proteins with overexpression of ChvI variants. Total proteins isolated from A. tumefaciens C58 strains containing the empty vector (V) or one of the plasmids expressing ChvI, ChvI(D52E) or ChvI(D52A) grown in AB-MES (pH 7.0) at 25°C for 6 h underwent western blot analysis with antibodies against ClpV, Hcp, and Atu4349. Protein levels of ClpV, Hcp, and Atu4349 were quantified with use of the UVP BioSpectrum 600 Imaging System and normalized to the level of endogenous RpoA. The level of vector control was set to 1. Data are mean ± SD of two biological replicates.(TIF)Click here for additional data file.Figure S5Alignment of the amino acid sequences of A. tumefaciens and S. meliloti ExoR. ExoR orthologs from Atume  and Smeli  were aligned with 74% identity. The identical amino acid residues are highlighted in black, and the arrow indicates a predicted signal peptide cleavage site. The amino acid residues used for mutagenesis are indicated.(TIF)Click here for additional data file.Figure S6Effect of trichloroacetic acid on migration of cellular and secreted Hcp proteins. (A) Total (T) and secreted (S) proteins were isolated from A. tumefaciens wild-type C58 grown in AB-MES (pH 5.5) at 25°C for 6 h. The supernatant was precipitated by TCA without washing or followed by 85% acetone wash. The proteins were analyzed by western blot analysis with antibodies against Hcp and ActC. The periplasmic protein ActC was an internal control. (B) Western blot analysis of total protein levels of Hcp or RpoA with or without 10% TCA.(TIF)Click here for additional data file.Figure S7Expression analysis of ChvG and ExoR driven by native promoters. (A) A. tumefaciens ΔchvG expressing pChvI-ChvG-HA driven by PchvIG native promoter on plasmid pRL662 was grown in AB-MES (pH 7.0 or pH 5.5) at 25°C for the indicated times. Total proteins were analyzed by western blot analysis with antibodies against HA (for detecting ChvG-HA) and RpoA. The positions of molecular mass markers (in kDa) are indicated on the left. (B) PCR confirmation of exoR-strep expressed from chromosomal locus. The wild-type exoR gene was replaced by the Strep-tagged exoR with confirmation by PCR analysis with primers corresponding to the exoR coding region and the downstream flanking region. A 130-bp product was identified in wild-type C58 (lane 1) and a 154-bp product in the strain with exoR-strep gene replacement (lane 2–5). (C) Western blot analysis of protein level of ExoR-Strep. Total proteins isolated from A. tumefaciens wild-type C58 strain and 4 colonies of the strain grown in AB-MES (pH 7.0) overnight at 25°C underwent western blotting with antibodies against strep (for detecting ExoR-strep) and RpoA. ExoR-Strep was detected from expression with the plasmid form (lane 6 ΔexoR containing pTrC-ExoR-Strep) but not from wild type C58 (lane 1) or the 4 independent colonies with chromosomal fusion .(TIF)Click here for additional data file.Table S1Bacterial strains and plasmids.(PDF)Click here for additional data file.Table S2Primers used in this study.(PDF)Click here for additional data file.