in contact with vancomycin (see figure 8C). Understanding this profile could be important for understanding vancomycin resistance, at least in SCO2594::Tn5062 mutant. We checked transcription of genes which could be implicated in vancomycin resistance, like for example; genes coding for sigma factors, penicillin-binding proteins, cell wall peptidoglycan biosynthesis enzymes (like femX [SCO3904] or mur genes or cseBC, coding for a two component system involved in cell wall stress sensing (Hong et al., 2002). The cseBC genes were not significantly regulated although they showed the same profile of whiB and van genes (see figure 8A); and the same happened with the only known target of WhiB in S. coelicolor, dpsA (Facey et al. 2011). In any case, only SCO1875, coding for a putative penicillin-binding protein, showed a significant regulation. This gene was more than two times up-regulated in the mutant strain. In a previous study, deletion of SCO1875 increased markedly vancomycin susceptibility in S. coelicolor; pointing to an important role of this penicillin-binding protein in glycopeptide resistance (Hesketh et al., 2011). The Pi cell wall content is highly reduced in the SCO2594::Tn5062 mutant According to Kleinschnitz and co-workers (2011), SCO2592 code for a B. subtilis tagE homologue; being its function the glycosylation of teichoic acids. Moreover, the transcription profile of SCO2592-94 resembles that of some B. subtilis teichoic acids genes (Liu et al., 1998). Therefore, we wondered if these genes could be implicated in the biosynthesis of any Pi polymer of the cell wall. To investigate this question parental and SCO2594::Tn5062 strains were cultured in the Pi-replete medium MG-18.5 and cell walls were isolated at the end of the exponential growth phase. The experiment clearly
Aqueous cell wall samples (200 µL) were applied over potassium bromide disks and dried at 50 ºC. Their infrared spectra were recorded by a Perkin-Elmer instrument with scanning range of 700–4200 cm−1 and resolution of 1 cm−1. RNA isolation and microarray hybridization Transcriptomic profiles of both S. coelicolor M145 and INB201 strains were determined using the data from the temporal series TS1 and TS3 of the ERA-NET SysMO project STREAM (GEO accessions GSE18489 and GSE31068). Experiments were performed in 3 liter fermentors as described by Nielset et al. (2010) and Thomas et al. (2012). For the transcriptomic analyses performed in this study, RNA samples from the liquid cultures were isolated after stabilization of 1 volume of culture with 2 volumes of RNA Protect Bacteria Reagent (Qiagen). The samples were treated with lysozyme (15 mg/mL), buffer RLT (Qiagen) and transferred to Lysing Matrix B tubes (MP Biochemicals) for mechanical disruption at Fast prep homogenizer (speed 6.5, 30 sec, Thermo Savant). The lysates were extracted with phenol-CIA and RNA was isolated from each sample using the RNeasy Mini Kit (Qiagen), including the recommended DNase digestion to eliminate chromosomal DNA contamination. The final elution step was carried out with 50 µL of nuclease-free water. RNA samples from the TSA cultures were purified as follows: the cells were carefully taken from the plates with a small scraper (previously disinfected with a NaOH-SDS solution and commercial RNaseZap purchased from Ambion) and immediately put in a FalcomTM tube containing glass balls and 5 mL of RNA Protect Bacteria reagent. Vigorous vortexing was carried out to provide disaggregation of the cells from the agar remnants and column