37% sodium bicarbonate and 10% fetal bovine serum at 37°C with 5% CO2. All work with live B. melitensis was performed in a biosafety level 3 laboratory at

Texas A&M University College Station, selleck kinase inhibitor per CDC approved standard operating procedures. All bacterialstrains used are listed in Additional File 1, Table S1. Generation of gene replacement and deletion mutants LuxR-like proteins were identified in B. melitensis using NCBI BLAST protein homology searches http://​www.​ncbi.​nlm.​nih.​gov/​. B. melitensis 16M luxR gene replacement and deletion mutations were created as previously described by our laboratory, with plasmids and strains generated described in Additional File 1, Table S1 and primers for PCR applications listed in Additional File 2, Table S2 [19]. For complementation of the ΔvjbR mutation, gene locus BMEII1116 was amplified by PCR primers TAF588 and TAF589, cloned into pMR10-Kan XbaI sites, and electroporated into B. melitensis 16MΔvjbR (Additional File 1, Table S1 and Additional File 2, Table S2). Gentamycin protection assay J774A.1 cells were seeded into 24-well plates at a density of 2.5 × 105 CFU/well and allowed to rest for 24 hours in DMEM. J774A.1 cells were infected selleck chemical with B. melitensis 16M or mutant strains in individual wells at an MOI of 20. Following infection, monolayers were centrifuged (200 × g) for 5 min and incubated for 20 minutes.

Infected monolayers were washed 3 × in Peptone Saline (1% Bacto-Peptone and 0.5% NaCl), and incubated in DMEM supplemented with gentamycin (40 μg/ml) for 1 hour. To collect internalized bacteria at time 0 and 48 hours post-infection, macrophages were lysed in 0.5% Tween-20 and serial dilutions were

plated to determine bacterial colony forming units (CFU). RNA collection Cultures were grown in Brucella Broth at 37°C with agitation. Cultures for the AHL experiments were grown with the addition of exogenous N-dodecanoylhomoserine lactone (C12-HSL, Rucaparib nmr Sigma, St. Louis, MO) added at inoculation (50 ng/ml) dissolved in DMSO (at a final concentration of 0.008%) [16]. Total RNA was extracted at mid-exponential (OD600 = 0.4) and early stationary (OD600 = 1.5) click here growth phases by hot acidic phenol extraction, as previously described [20]. Contaminating DNA was degraded by incubation with DNAseI (Qiagen, Valencia, CA) following manufacturer’s instructions and purified using the HighPure RNA isolation kit (Roche, Indianapolis, IN). RNA integrity, purity and concentration were evaluated using a 2100 bioanalyzer (Agilent, Santa Clara CA), electrophoresis, and the Nanodrop® ND-1000 (Nanodrop, Wilmington, DE). DNA and RNA labeling for microarrays B. melitensis 16M genomic DNA was processed into cDNA using the BioPrime® Plus Array CGH Indirect Genomic Labeling System (Invitrogen, Carlsbad, CA) and purified using PCR purification columns (Qiagen, Valencia, CA) following the manufacturer’s instructions and eluted in 0.1× of the supplied elution buffer.