, 2005). The function of this gene is not known. In other bacterial species that possess more than one chaperonin gene, the differential expression of these genes is generally seen. In particular, in cases where one gene has been shown from genetic analysis to be the essential chaperonin, this gene generally shows the highest level of expression, whereas the other genes that may play additional roles are expressed at lower levels or under more specific conditions (e.g. Fischer et al., 1993; de León et al., 1997; Kovács et al., 2001; Gould et al., 2007; Hu et al., 2008; Sato
et al., 2008). As part of our characterization of the three chaperonin genes and the proteins that they encode in the mycobacterial species M. smegmatis, we have measured their expression under normal growth and in response to various stresses, and selleck compound we report these results here. The bacterial strains are shown in Table 1. All oligonucleotides were synthesized by Alta Biosciences or [for use in quantitative real-time PCR (qRT-PCR)] by Applied Biosystems, and are shown in Table 2. Escherichia coli was grown in Luria–Bertani
(LB) broth. A solid medium was prepared by adding 1.5% agar to the LB broth. Mycobacterium smegmatis was cultured in Difco Middlebrook 7H9 learn more broth (BD Biosciences) containing ADC and 0.05% Tween 80, or in Difco Middlebrook 7H10 agar with ADC (BD Biosciences) and 0.05% Tween 80. Antibiotics were used at 100 μg mL−1 (ampicillin) or 50 μg mL−1 (kanamycin) for E. coli, and 20 μg mL−1 (kanamycin) and 150 μg mL−1 (hygromycin) for M. smegmatis. Protein sequences were identified and extracted from GenBank, aligned using clustalw with
default values, and phylogenetic trees were drawn using phylip or neighbourhood joining, using upgma for clustering. A 10 mL mid-log culture of M. smegmatis (grown in 7H9 Niclosamide and ADC with 0.05% Tween80) was mixed with 4 vol. of 5 M GTC buffer (5 M guanidinium isothiocyanate) lysis solution and mixed rapidly by swirling. Cells were pelleted by centrifugation at 1200 g for 30 min, resuspended in 1 mL of 4 M GTC solution, centrifuged for a minute at 16 000 g and resuspended in 1.2 mL of TRI reagent (Fluka Biochemicals), which was added to 0.5 mL of 0.1 mm ceramic beads in 2-mL screw-capped microcentrifuge tubes. The tubes were spun using a reciprocal shaker (Hybaid Ribolyser) at the maximum speed setting (6.5) for 45 s, and then left at room temperature for 10 min. Chloroform (200 μL) was then added and the tubes were vortexed for 30 s. The tubes were then left at room temperature for 10 min to partition the aqueous and the organic phases and then centrifuged at 16 000 g at 4 °C for 15 min. The lighter aqueous phase was transferred to a fresh tube, mixed with an equal volume of chloroform, vortexed and incubated at room temperature for 10 min before centrifuging at 16 000 g at 4 °C for 15 min. The aqueous phase was transferred to a new tube and 0.8 vol.