The culture was incubated at 22°C for 48 h with orbital shaking. RNA was isolated from the bacterial culture with a commercial NucleoSpin RNA Plant kit (Macherey-Nagel GmbH & Co. KG, Germany). The RNA concentration was determined using a Nanodrop ND-1000 (NanoDrop Technologies Quizartinib molecular weight Wilmington, DE) and was optimised up to 50 ng/μl for RT-PCR assays and 1 μg/μl for Northern blotting. The integrity of the RNA sample was assessed by agarose gel electrophoresis. RT-PCR was performed using 100 ng of RNA at a final volume of 50 μl using the Titan OneTube RT-PCR system, according to the manufacturer’s instructions (Roche Diagnostics). The primers were

designed by using sequences located between each gene (Additional file 2: Table S1). A 40-cycle amplification programme (94°C for 30 s, 58°C for 1 min, and 68°C for 1 min) was performed followed by a final extension cycle at 68°C for 7 min. Positive control reactions that contained DNA

isolated from each corresponding bacterial strain were included in Selleck GW786034 all assays. Northern blotting was performed using a denaturing agarose gel (0.7%) and formaldehyde (2.2 M). The samples were prepared with 20 μg of total RNA in MOPS running buffer with 2.2. M formaldehyde and 50% formamide and denatured at 65°C for 10 min. The agarose gel was run for 90 min at 60 V. The RNA was transferred to a nylon membrane by capillary diffusion using 10× saline-sodium citrate buffer (SSC) and was immobilised by UV cross-linking. The hybridisation was performed with radioactively labelled probes (dCTP32). Characterisation of the mgo operon promoter We used pMP220 [30] as the promoter-probe vector Tenofovir mouse to measure transcriptional activity by β-galactosidase (β-Gal) expression. The amplicon (1008 bp), which included the putative promoter region upstream of mgoB, was cloned into the multicloning site using the EcoRI and PstI restriction sites, which were not present in the cloned sequence. The resulting plasmid, pMPmgo, was transformed into multiple bacterial species (Table 5), and β-Gal assays were performed [17, 18]. The protocol followed the assay Ro-3306 manufacturer described by J.H. Miller

[18], except for the addition of an extra step. In our assay, the cells were pelleted and then resuspended in assay buffer to eliminate any error in the detection of β-galactosidase enzyme activity due to the effects of different carbon sources present in the growth medium. Additionally, 5′-RACE (Rapid Amplification of cDNA Ends) experiments were performed to locate the +1 nucleotide in the mgo operon transcript and determine which putative promoter is active during mgo operon transcription. The commercial SMART™ RACE cDNA Amplification Kit (Conthech Laboratory, Inc.) was used. Moreover, mRNA from UMAF0158 was obtained by a commercial NucleoSpin RNA Plant kit (Macherey-Nagel GmbH & Co. KG), as described above. Extract complementation Extracts from wild-type UMAF0158 and the mutant UMAF0158ΔmgoA were used in the complementation experiments.