The new construct, pER164 (Fig. 1), was conjugated into B. fragilis 638R and IB263 strains by triparental mating to construct BER-96 and BER-105, respectively. For the microscopy slides, 1 mL of bacterial cultures

grown to mid-log phase in BHIS under conditions described above were centrifuged at 3000 g for 3 min and washed once in 1 mL of phosphate buffer saline (PBS) I-BET-762 cost (4.3 mM dibasic sodium phosphate, 1.47 mM monobasic potassium phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride, pH 7.4). Bacteria were suspended in 1 mL PBS and a drop of this suspension was added to each slide and allowed to air-dry. The coverslips were mounted with glycerol and the slides were analyzed with a Confocal Microscope Zeiss LSM 510 using an excitation of 450 nm selleck products and an emission filter in the range of 475–525 nm. For dual channel fluorescent color detection in slides stained with Alexafluor-546-phalloidin

conjugate (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction, an excitation at 556 nm and emission at 573 nm were also used. The J774.1 macrophage cell lines was grown in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum and 2 mM l-glutamine. The cells were grown over sterile coverslips placed inside six-well microplates at 37 °C in a 5% CO2 humidified atmosphere. For all assays, six-well plates were seeded with approximately 2 × 105 macrophages mL−1 and incubated until confluence was reached. The bacterial cell numbers were determined spectrophotometrically at 600 nm. The assay was carried out by inoculating B. fragilis at an approximate multiplicity of infection of 100 into the six-well plates under anaerobic conditions. Infected monolayers were incubated for 1 h Exoribonuclease inside the anaerobic incubator to allow phagocytosis and internalization to occur. Then, the monolayers were

washed three times with PBS without an antibiotic to remove unbound bacteria. The cells were then fixed with 3.7% formaldehyde for 10 min and washed three times with PBS. Macrophages were stained with Alexafluor-546-phalloidin conjugate. The coverslips were removed from the wells and placed on top of glass slides for laser confocal microscopy analysis as described previously. In this study, we show the use of the fluorescent protein BS2 as a reporter for in vitro and in vivo gene expression studies in the anaerobe B. fragilis. When the promoterless bs2 gene was cloned in fusion with the starch/maltose and oxygen inducible promoter osu (Spence et al., 2006), addition of maltose was able to induce expression of fluorescent BS2 under anaerobic conditions compared with uninduced culture controls. These results clearly demonstrate that expression of BS2 in cultures of B. fragilis BER-85 in the absence of oxygen yield an intense fluorescence characteristic of BS2.