Macrophages were seeded in 75 cm2 culture flasks (BD Falcon) 20 hours before infection. P. aeruginosa cells were grown in LB up to an OD600 of 1.0. The J774 macrophages (1.8 × 107 per flask) were infected with bacteria at a multiplicity of infection of 10 for 1 or 2 hours. The supernatants were then withdrawn
and the non-phagocytosed bacteria were www.selleckchem.com/products/dinaciclib-sch727965.html harvested by centrifugation prior to RNA purification. In semi-aerobic growth conditions, overnight P. aeruginosa cultures were diluted to OD600 0.075 in LBN (LB with NaCl 2.5 g/L and KNO3 1%) into medium-filled flasks plugged with non-porous caps. The medium was saturated with N2 gas by bubbling for 30 min, and the cultures were grown with agitation at 37°C. To study the impact of the carbon or nitrogen source on fdx1 expression, P. aeruginosa was grown in minimal M63 medium supplemented with 0.5% casamino-acids selleck compound and with either 40 mM glucose or pyruvate, or with 15 mM ammonium or 40 mM nitrate, as carbon and nitrogen sources,
respectively. Growth with p-hydroxybenzoate Methane monooxygenase as carbon source was carried out in the synthetic medium described for bacteria degrading aromatics in the
absence of oxygen . Construction of lacZ reporter insertion PCR amplification was used to produce the two fdx1 promoter fragments: primers FDX-Eco and FDX-Bam (Table 1) amplified a 555 bp fragment, and primers FDX-Eco200 (Table 1) and FDX-Bam a 237 bp fragment. The PCR products were ligated into the pCR-Blunt II-TOPO vector (Invitrogen) and sequenced. The 0.55-kb and 0.24-kb fragments were transferred into mini-CTX-lacZ , providing the pCTX-pFdx1Z and pCTX-pFdx1shortZ plasmids, respectively. The plasmids were introduced into P. aeruginosa by triparental conjugation, using the conjugative properties of the helper plasmid pRK2013 . The transconjugants were selected on PIA plates containing tetracycline: plasmids were inserted at the chromosomal ϕCTX attachment site (attB site). The pFLP2 plasmid was used to excise the Flp-recombinase target cassette as described . The corresponding P. aeruginosa strains were designated with the pFdx1Z and pFdx1shortZ extensions. Table 1 Oligonucleotides used in this work.