The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells. ClpV is usually a member of the AAA+ (ATPases associated with various cellular activities) protein family (8, 11). It forms oligomeric complexes to energize the system for the secretion of effector protein, which include the secreted VgrG (valine glycine repeat) and Hcp (hemolysin-coregulated protein) protein (1, 12,C17). It has been reported that the T6SS contributes to the virulence development of various pathogens and is usually often activated upon contact with target cells for the secretion of effector proteins (1,C7, 14,C17). The alpha-Cyperone manifestation and assembly of the T6SS are tightly controlled at both the transcriptional and posttranscriptional levels (12, 13, 18). Furthermore, the IcmF protein of the T6SS has been shown to be involved in flagellar rules and affects motility and biofilm formation (15). The flagellum is usually composed of a basal body, a connect, and a filament. FliC, a component of the filament, is usually transported from the cytoplasm by a number of transport systems in different bacterial species, including the SPI1 type III secretion system (T3SS), the Dot/Icm type IV secretion system (T4SS), and the locus of enterocyte effacement (LEE)-encoded T3SS in serovar Typhimurium, is usually considered a commensal of the intestinal tract in humans and other animals (21). However, can also cause diarrhea and other infections in humans (22,C25). Relatively little is usually known about the virulence of strain, CF74, that showed an aggregative adherence pattern and cytotoxicity to HEp-2 cells. The strain was found to contain a complete T6SS located on a genomic island (GI), and the T6SS gene cluster consists of 16 genes, including key T6SS genes (strain CF74 was isolated from a fecal sample from a goat, as reported previously (26). All strains were produced aerobically at 37C in Luria-Bertani alpha-Cyperone (LB) medium. Antibiotics were added at the following concentrations: 100 mg/ml for ampicillin and streptomycin and 30 mg/ml for chloramphenicol where appropriate. All strains and plasmids used in this study are listed in Table 1. TABLE 1 Strains and plasmids Construction of isogenic mutants and plasmids. All in-frame deletion mutants were generated in strain CF74 via double crossover using the suicide plasmid pWM91 as described previously (27, 28). The upstream and downstream regions of the gene of interest were amplified using the primers shown in Table H1 in the supplemental material. Using fusion PCR of these two fragments, we generated a fragment that was cloned into pWM91, a plasmid made up of the counterselectable gene. The recombinant plasmids were then purified and introduced into SM10 and conjugally transferred into CF74, and deletion of the genes of interest was selected in LB agar with 10% sucrose and without NaCl. Chromosomal deletion mutants were identified by colony PCR and quantitative reverse transcription (qRT)-PCR. CF74 deletion mutants were complemented by pBAD24 harboring the genes of interest, allowing arabinose-controlled gene manifestation. For transformation of plasmids into CF74, electrocompetent cells were prepared as described previously (29). RNA extraction and qRT-PCR. To prepare cells for RNA extraction, new LB medium was inoculated from an overnight culture of CF74, deletion mutants, or their complementation mutants (1:100) and incubated at 37C with shaking at 220 rpm. The strains were collected at an optical density at 600 nm (OD600) of 1.0. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. alpha-Cyperone RNA samples were further purified using Rabbit polyclonal to AARSD1 the RNeasy minikit (Qiagen, Valencia, CA), followed by treatment with DNase I (Qiagen, Valencia, CA) to eliminate genomic DNA contamination. The RNA size, honesty, and total amount were assessed using a alpha-Cyperone Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). To measure gene transcription in different strains, qRT-PCR was performed using specific primers (see Table H1 in the supplemental material) based on the targeted genes. Total RNA (1.0 mg) was reverse transcribed to generate cDNA as the template for qRT-PCR. qRT-PCR was carried out using SYBR Premix Ex lover Taq II (Perfect Real Time; TaKaRa) using a Rotor-Gene Q thermal cycler (Qiagen, Valencia, CA). Data were analyzed with Rotor-Gene Q series software version 1.7 (Qiagen, Valencia, CA). The data were normalized to the endogenous.