Besides sporulation, can undergo a differentiation process in which short swimmer cells become elongated and hyperflagellated swarmer cells that favor migration of the bacterial community on a surface. in is a Gram-positive, motile, spore-bearing rod, frequently isolated from the soil, where the spore ensures its persistence under adverse conditions. Long known as agent of food-borne diseases, this organism is now recognized to be able to cause local and systemic infections in humans (Bottone, 2010; Logan, 2012; Celandroni et al., 2016). The pathogenic potential of this bacterium is related to the secretion of several virulence proteins, e.g., hemolysins, phospholipases, trimeric toxins (hemolysin BL, HBL; non-hemolytic enterotoxin, NHE), cytotoxin K (CytK), proteases (Senesi and Ghelardi, 2010; Ramarao and Sanchis, 2013; Je?berger et al., 2015), and to motility modes, such as swimming and swarming (Senesi et al., 2010; Celandroni et al., 2016). Bacterial swarming is a flagellum-driven social form of locomotion in which cells undergo a periodical differentiation process leading to the production of long and hyperflagellated elements, the swarmer cells, which coordinately migrate across surfaces (Kearns, 2010; Partridge and Harshey, 2013). Swarming confers an advantage for the colonization of natural and host surfaces and can contribute to bacterial virulence. Notably, swarming increases HBL secretion by (Ghelardi et al., 2007) and enhances the pathogenicity of this bacterium in an experimental endophthalmitis model (Callegan et al., 2006). In a previous study, we demonstrated that the protein FlhF plays a major role in controlling the arrangement of flagella in (Salvetti et al., 2007). The proteins FlhF and FlhG are essential for establishing correct place and quantity of flagella in many but not all bacterial species (Schniederberend et al., 2013). Telatinib In (Zanen et al., 2004). Differently, in and mutant of showed an increase in the extracellular levels of NHE and a decrease in HBL and phosphatidyl-choline specific phospholipase C (PC-PLC) (Salvetti et al., 2007). Thus, the aim of the present study was to gain more insight into the function of FlhF in by evaluating the effects of FlhF depletion on interconnected cellular functions such as swarming, protein secretion, and virulence, which may all Telatinib depend from protein targeting to the membrane. Materials and Methods Bacterial Strains and Growth Conditions ATCC 14579 wild type (wt), its (GeneBank ID: dependent gene expression. Analysis BLAST1 was used for comparative analysis of nucleotide and protein sequences. Protein sequences in the FASTA format were retrieved from the UniProt database2 (The UniProt Consortium, 2015). Functional domain analysis was performed using the ProDom Server3 (Bru et al., 2005). The presumptive secondary and tridimensional structure of proteins were Rabbit Polyclonal to IL4 generated using the Phyre2 web portal for protein modeling, prediction and analysis4 (Kelley et al., 2015) and the Raptor X Structure Prediction Server5 (K?llberg et al., 2012), respectively. Swarming Motility For each experiment, swarm plates (TrA plates; 1% tryptone, 0.5% NaCl, 0.7% granulated agar) were prepared fresh daily and allowed to sit at room temperature overnight before use (Salvetti et al., 2011). Swarming was initiated by spotting 50 l of a culture containing approximately 2104 cells/ml onto the center of TrA plates, and incubating cultures at 37 C. Swarming migration was evaluated by measuring colony diameters after 8 h. Since flagella are very fragile, bacterial samples were taken by slide overlay of single agar blocks (5 mm 5 mm) that contained different colony portions. Bacterial cells were stained with tannic acid and silver nitrate (Harshey and Matsuyama, 1994) for microscopy. Several samples were analyzed at 1000 magnification using an optical microscope (BH-2; Olympus, Tokyo, Japan). All experiments were performed in duplicate in three separate days. Preparation of Culture Supernatants Protein samples were prepared by growing bacterial cells to the late exponential growth phase in BHIG at 200 Telatinib rpm for 6 h at 37 C. Culture supernatants were collected by high-speed centrifugation (10000 and molecular weight (Mw) compared to the approximate experimental values observed on 2-DE gels. Identified proteins were classified based on their biological functions using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database resource7. Protein sequences were analyzed using the SIGNALIP 4.1 Server8, TATP 1.09, SecretomeP 2.0 Server10.