While the G2 PhyloChip is an excellent tool for identifying known bacteria, it contains only 300 archaeal sequences, which were not utilized because bacterial-specific primers were used. Furthermore, there is currently no microarray that is designed to identify protozoa or fungi. Next generation (high-throughput) sequencing is needed to validate the bacterial population findings of the present study, as well

as identify the protozoal, archaeal and fungal populations present in the moose rumen. The PhyloChip, like all methods that do not rely on culturing, cannot be used to differentiate between transient and colonizing species. It can be assumed that some species found in the moose are simply passing through the digestive tract, having been picked up from the environment, and are not colonizing the tract. Despite

this, CAL-101 supplier these transient www.selleckchem.com/products/acalabrutinib.html bacteria may still have an impact on the dynamics within the rumen, and it is important to take a holistic approach when looking at mixed environmental samples. It is also possible that some of these unclassified bacteria which are presumed transient, such as the soil or water clones, are actually colonizing the moose digestive tract and are simply unique to moose. Methods Sample collection All samples were obtained with permission of licensed hunters through the Vermont Department of Fish and Wildlife. Whole rumen (R) and colon (C) contents were collected from moose shot during the October 2010 moose hunting season in Vermont. Samples were collected by hunters within 2 h, if not sooner,

of death and put on ice immediately. Hunters were given a written set of instructions about sample collection, and had been instructed verbally as well, to fill the collection containers with material taken buy Palbociclib from well inside the rumen and colon, and to seal the container quickly to minimize overexposure to oxygen. Samples were then transferred to the laboratory within 24 h, and stored at −20°C until DNA extraction. A total of eight rumen and six colon samples (Table 3) were collected from eight moose. Twelve of the samples were paired rumen and colon contents from the same animal, and two rumen samples did not have corresponding colon samples. Moose were weighed and aged, by examining the wear and replacement of the premolars and molars of the lower jar, by Vermont Fish and Wildlife biologists at the mandatory reporting stations. Table 3 Statistics for samples taken from moose shot in October 2010 in Vermont during the moose hunting season Moose Sample location Sample name Gender Weight, dressed carcass (kg) Approx. age (yr) 1 Rumen 1R F 185 1   Colon 1C       2 Rumen 2R F 244.55 3   Colon 2C       3 Rumen 3R M 186.36 2   Colon 3C       4 Rumen 4R M N/A N/A 5 Rumen 5R M 319.09 4 6 Rumen 6R F 259.55 3   Colon 6C       7 Rumen 7R M 301.36 4   Colon 7C       8 Rumen 8R M 405.

By varying magnetic field direction in or out of the sample plane, we observed linear and quadratic magnetic field dependence of the photocurrents, respectively. More information about excitation and relaxation of electrons in this structure were

obtained from the experiments. Methods The InAs/GaSb superlattice was fabricated by molecular beam epitaxy technique on semi-insulating (001)-oriented GaAs substrate. The 500-nm GaAs and 1,000-nm GaSb buffers were deposited on the substrate to relieve the lattice mismatch. Then an InAs/GaSb superlattice of 155 periods was deposited. The monolayer thicknesses of InAs and GaSb are 3.85 and 2.60 nm, respectively. The sample was not intentionally doped. The Lapatinib energy gap of this structure calculated by the k ·p theory is 129.5 meV. The standard Hall measurement demonstrates that the sample is n-type at room temperature, i.e. electrons are the main carriers contributing to transport. Since in the n-type superlattice spin relaxation time and lifetime of holes are much shorter than

those of electrons, we neglect the contribution of holes to the magneto-photocurrents. Four pairs of ohmic contact electrodes which are parallel to [1 0], [110], [100] and [010] crystallographic directions were equidistantly made on the edges. The experimental setup is shown in Figure 1b. A linearly polarized 1,064-nm laser normally irradiated on the center of the sample to excite direct interband transition of electrons. Hence, Gefitinib clinical trial the circular photogalvanic effect and linear photogalvanic effect [3] are forbidden in this C 2v symmetry structure for the normal incidence case. A permanent magnet was used to generate magnetic field which can be along arbitrary direction Urease in the sample plane. The investigation of photogalvanic effect was carried out at room temperature by rotating the magnetic field. The data were collected by a standard lock-in amplification technique. Specifically, the laser power was about 63 mW, the light spot diameter was 1.2 mm and the permanent magnet strength was 0.1 T. Besides, we choose x, y and z to be along [1 0], [110] and [001] crystallographic directions,

respectively. Results and discussion In-plane magnetic field-dependent MPE As shown in Figure 2, by rotating the magnetic field in the x-y plane, the MPE currents in [1 0], [110], [100] and [010] crystallographic directions were detected. The current, as a function of φ, can be simulated by the combination of sinφ and cosφ no matter which pair of electrodes are chosen. They reach the maximum when the magnetic field is perpendicular to the detected direction and the minimum when the magnetic field is paralleled to the detected direction. Figure 2 The currents in [010], [1 0], [100] and [110] crystallographic directions when the linearly polarized direction of the incident light is along [110] crystallographic direction.

The V th is defined as the gate voltage at I d = 10−9 A. The temperature coefficients of V th are −1.34 and −5.01 mV/°C for GAA and planar find more JL TFTs, respectively. According to [13], the variation of in n-type JL devices can be expressed as follows [13]: (4) Figure 4 Impact of temperature dependence on the (a) V th and (b) on-state currents. For JL GAA TFTs (L g = 1 μm, 60 nm) and JL planar TFTs (L g = 1 μm). The Vth and Ion for JL GAA TFTs are less sensitive to temperature than JL planar TFTs. where V fb is the flat-band voltage, C ox is the gate oxide capacitance per unit length, A is the device cross-sectional area and P is the gate perimeter. The first term in the right side of Equation

4 is depended on the flat-band voltage variation with temperature. For N D = 1 × 1019 cm−3, the value of is approach to −0.49 mV/°C as the devices in [13], which has a P+ polycrystalline silicon gate and the same doping concentration. The second term

represents the effect of incomplete ionization. The doped impurities are almost completely ionized at those temperatures higher than room temperature. Thus, the doping concentration variation with the temperature has a slight dependence on temperature. The third term, depending on the electron effective mass, also has a smaller dependence on T than the other terms. The theoretical value of is about −0.49 mV/°C; although the of −1.34 mV/°C in JL GAA TFTs is larger than theoretical value, but is comparable with current SOI-based JNT ( approximately −1.63 mV/°C) [7] due to the use of the multi-gate structure and formation of

a crystal-like nanosheet Src inhibitor channel with fewer traps by oxidation process. Therefore, JL TFTs with the GAA structure and ultra-thin channel shows an excellent immunity to the temperature dependence on V th and competes with SOI-based JNT. Figure 4b presents the measured on-current (I on) as a function of temperature. The I on is defined as the drain current at V g = 3 V for JL planar TFTs and at V g = 6 V for JL GAA TFTs. The JL GAA TFTs show a slightly better I on variation with Etofibrate temperature than the planar ones, possibly owing to a smaller in JL GAA TFTs. Conclusion This work has presented a high-temperature operation of JL TFTs. The high temperature dependence of JL GAA and planar TFTs is also studied. The variation of parameters such as V th, I on, SS, and I off are analyzed as well. The variation of the SS with temperature for JL GAA TFTs is close to the ideal value (0.2 mV/dec/K) owing to the ability of the oxidation process to form a nanosheet channel and crystal-like channel. Additionally, I off is negligibly small for JL GAA TFTs, owing to quantum confinement effect; its E g of 1.35 eV is also extracted. The JL GAA TFTs have a smaller than that of JL planar TFTs owing to the GAA structure and ultra-thin channel. Moreover, the measured of JL GAA TFTs competes with that of SOI-based JNTs.

The signaling cascade is mainly initiated by binding of M. avium components Sirolimus in vivo to TLR2 followed by recruitment of the MyD88 adaptor molecule and the activation of NFκB and MAP kinases. This chain

of events ends with the induction of inflammatory cytokines [10] controlling macrophage activation and granuloma formation. We monitored the induction of cytokine expression of THP-1 macrophages by the WT and the mutants in order to evaluate their ability to stimulate the immune signaling. To this aim we quantified the secretion of selected cytokines: the pro-inflammatory cytokines TNF-α, IL-1β and the anti-inflammatory cytokine IL-10. Five independent experiments were normalised for WT (expression ratio 1) to determine the expression ratio for the mutants in comparison to WT. While results for TNF-α and IL-1β were not significantly different as compared to WT, IL-10 was significantly (P <0.007) up-regulated for mutant MAV_4334 (Figure  5). IL-10 can inhibit the production of inflammatory cytokines such as TNF-α in monocytes pre-activated by IFN-γ and LPS [67, 68] and therefore plays an important role in the immune response. Figure 5 Induction of IL-10 cytokine secretion

by infected macrophages. THP-1 cells (2.0×105) were infected (MOI 50) with mutants and WT. After 24 hours cytokines from supernatants were measured by ELISA. When compared to BMS 907351 WT a P value <0.01 (two-tailed, unpaired Mann–Whitney test) was considered very significant (**). Intracellular survival The ability to survive and even replicate inside the phagosomes of macrophages is an important virulence factor of mycobacteria and was therefore included in our screening options. Infection experiments with macrophages give information on the early host response to mycobacterial infections [69]. Different types of macrophages

or monocytic cells have been employed to assess mycobacterial virulence and among these the human macrophage-like cell line THP-1 has proven a suitable system for virulence testing [69, 70]. It was shown that THP-1 cells are similar to primary human monocyte-derived macrophages with respect to their ability to take up mycobacteria and limit their growth [71]. We infected THP-1 cells that had been differentiated by PMA with the WT and the mutants. Intracellular Chlormezanone mycobacteria were measured by quantitative real-time PCR and CFU by plating. Survival of mutants in THP-1 cells was not consistently different if compared to the WT (data not shown). More significant differences were obtained when using human blood monocytes for the infection experiments. The growth of mutant MAV_4334, MAV_1778 and MAV_3128 was affected the most in human monocytes (Figure  6). They were reduced significantly for the first two days (P < 0.05 to P < 0.01). Mutant MAV_4334 and MAV_1778 (Figure  6 A and C) were almost reduced to half during the first two days.

Due to their excellent mechanical stability, high conductivity, and antifouling properties, CNTs have been widely employed for GOx immobilization in biosensors [15]. Moreover, the CNT platform provides a more appropriate environment for immobilized GOx and therefore provides a quick shuttling of electrons with the surface of an electrode [15, 16]. In sensor technology, analytical modeling based on experimental finding is still ongoing. This study proposes an analytical glucose biosensor model of single-wall carbon nanotube field-effect transistor

(SWCNT 5-Fluoracil manufacturer FET) to predict the drain current versus drain voltage (I-V) performance. For the first time, the effects of glucose adsorption on CNT electrical properties, namely gate voltage, are studied and formulated versus a wide range of glucose concentration. Methods Sensing mechanism In this section, the methods of immobilization will be described to explain the sensing mechanism of a biosensor. Immobilization is a process to integrate a biocatalyst with a matrix that it is not soluble in aqueous media. A wide variety of approaches can be

applied for the immobilization of enzymes or cells on a variety of natural and synthetic supports. H 89 Both of the immobilization approach and support are dependent on the type of enzyme and substrate [17, 18]. Enzymes are very instable and sensitive to their environment [19]. When no special precaution is required, some common approaches, such as deactivation on an adsorption and chemical or thermal inactivation, are adopted [19, 20]. The important techniques that maintain the enzyme activity of immobilization are encapsulation, covalent immobilization, and site-specific mutagenesis [15, 21]. Ultimately, the application of the new materials will generally affect the quality of the sensing mechanism. Because of the high surface

area-to-volume ratio, CNTs demonstrate good device performance [22] when they are used as a semiconducting channel in biosensors [23]. The CNT application on glucose detection has been experimentally reported in [24] where GOx is utilized as an enzyme. The fabrication process of the SWCNT-based (1 to 2 nm in diameter, 50 μm in length) [25, 26] electrochemical glucose Ribonucleotide reductase biosensors using GOx [24] is depicted in Figure 1a,b. Polyelectrolytes, such as poly(diallyldimethylammonium chloride) (PDDA) and polystyrenesulfonate (PSS) are implemented [24]. Figure 1a shows the assembly of PDDA/SWCNT on polyethylene terephthalate (PET) polyester flexible substrate, and GOx biomolecular assembly is depicted in Figure 1b. Figure 1 Schematic fabrication process and a field-effect sensor. (a) Schematic fabrication process of glucose sensor [24]. (b) Proposed combination of metal electrodes made of chromium or gold, a layer of GOx biomolecular assembly, and SWCNT channel in the form of FET. To produce stable negative charges, GOx is dissolved into a phosphate-buffered saline (PBS) with a concentration of 1 mg/mL.

RNA extraction and cDNA synthesis Total RNA was prepared using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. RNA was treated with RNase (Invitrogen) in the presence of 50 μM T7 (dT12) AP1, T7 (dT12) CT99021 AP5 and T7 (dT12) AP8 primers in 20 μl RT buffer (1× Superscript II RT buffer, 10 mM DTT, 0.025 mM dNTP), at 25°C for 5 minutes, followed by 50°C for 50 minutes. Reverse transcriptase was inactivated at 70°C for 15 minutes. Differential display and full-length

gene cloning Differential display was performed using Hieroglyph mRNA Profile Kit (Beckman, CA, USA). Briefly, PCR amplification was done using 1.5 μl of the cDNA, primed with arbitrary P primer and anchored T primer. Amplification at (95°C 2 minutes) 1 cycle, find more (92°C for 15 seconds, 50°C for 30 seconds, 72°C for 2 minutes) 4 cycles, (92°C for 15 seconds, 60°C for 30 seconds, 72°C for 2 minutes) 30 cycles, followed by a final extension at 72°C for 7 minutes on a GeneAmp PCR system 9600 (Perkin-Elmer, Norwalk, USA). Following amplification of randomly primed mRNAs by RT-PCR, the cDNA products were heated at 95°C for 2 minutes and separated on a denaturing 5.6% polyacrylamide gel at 55°C for 5 hours using

a Genomyx LR DNA Sequencer (Beckman), under 3000 V. Bands exclusively present in either of two samples were considered as candidates of differentially expressed transcripts, which were excised, eluted, re-amplified, and subcloned into the T easy vector (Promega, San Luis Obispo, CA, USA). The sequence reactions were performed by Invitrogen. Sequence homology to published database was analyzed with the BLAST program at the internet site of NCBI (National Center for Biotechnology Information). 5′-RACE (rapid amplification

of cDNA 5′ ends) and 3′-RACE were used to isolate the complete cDNA. The human Marathon-ready cDNA (Clontech, Heidelberg, Germany) served as the template. Real-time quantitative reverse transcription polymerase chain reaction We measured LCMR1 gene expression in 95C and 95D cell lines by real-time quantitative RT-PCR in an ABI PRISM 7500 Sequence Detection System. The real-time RT-PCR allows, by means of fluorescence emission, the identification of the cycling point when PCR product is detectable. The Ct value inversely correlates with the starting all quantity of target mRNA. Measurements were performed in duplicate and the controls were included in which the reaction mixture contained no cDNA. The amount of target mRNA after normalized to the loading control β-actin was calculated by the Ct method. Primers for β-actin and LCMR1 mRNAs were chosen using the Primer Express 2.0 software (Applied Biosystems, Foster City, USA). Primers for LCMR1 were: 5′-AACAGAGCCGTACCCAGG AT-3′ (Forward) and 5′-GGGTGGTCTGGACATTGTC -3′ (Reverse). Primers for β-actin were: 5′-CATGTACGTTGCTATCCAGGC-3′ (Forward) and 5′-CTCCTTAATGTCACGCAC GAT- 3′ (Reverse).

The induction of hrp genes

in bacteria occurs soon after the first contact with plant tissue. Expression of hrp genes are detected as early as 1 h after inoculation and continue NVP-BGJ398 cost to increase for at least 6 h [6]. However, no specific plant-derivatives have been identified as inducers of hrp genes, and in Ralstonia solanacearum some evidence suggests that the full induction of hrp genes requires contact with plant tissues [7]. The hrp genes are also induced in vitro when bacteria are grown in minimal medium with carbon sources such as sucrose, fructose or mannitol, low pH and a low N/C ratio [6]. Minimal media with these characteristics seems to mimic some of the conditions bacteria might find Ku-0059436 mouse within the apoplast. It has been suggested that the induction of hrp genes after contact with plant tissues could result from alterations in the nutritional status of the bacteria [2, 6]. During the interaction with their host, it is thought that bacteria commonly detect specific plant metabolites, which are used as signals for changing their gene expression patterns, allowing them to adapt to the plant environment. Specific plant molecules such as phenolic β-glycosides, shikimic and quinic acids, and pectin oligomers

have been reported to activate the expression of genes involved in toxin synthesis and cell wall degradation [8–10].

In this study, we used microarray analysis to identify genes of P. syringae pv. phaseolicola NPS3121 differentially expressed in response to metabolites present in plant tissue extracts [11]. Bacteria were grown on minimal medium supplemented with bean leaf extract, apoplastic fluid or bean pod extract. By using these three types of extract, we were able to identify Idoxuridine bacterial genes that possibly facilitate the colonization of susceptible plant tissues, such as bean leaves and/or apoplastic fluid which are known targets during the infection process of P. syringae pv. phaseolicola NPS3121 [11, 12]. Results and Discussion Leaf extracts and apoplastic fluid produce highly similar transcriptional responses We decided to test bean leaf and pod extracts and apoplastic fluid since these are thought to be the primary environments that P. syringae pv. phaseolicola encounters during infection, and in which nutrient assimilation, plant signal recognition and stress responses can occur [13, 14, 1, 12]. To this end, P. syringae pv. phaseolicola NPS3121 was grown at 18°C in M9 minimal medium with glucose as a carbon source. When cultures reached the mid-log phase (OD600 nm 0.6) bean leaf extract, apoplastic fluid or bean pod extracts were added to a final concentration of 2% and an equal amount of minimal medium was added to a control culture.

Apparently, nitric acid

content influenced the morphology, giving spheres as the prevailing output. No correlation was observed between the acid content and sphere size, but it apparently affected the rate of condensation and thus the spherical texture. When employing sulfuric acid (SA), multishapes were seen both at 1 SA and 2 SA (see Figure 5). Regardless of the content, a nonuniform mix of shapes was obtained including spheres (solid and hollow), small fibers, and whirling rods. At a higher molar ratio (3.34 SA), no product was obtained, suggesting that at high sulfuric acid ratios, the growth becomes extremely slow. Figure 4 SEM images of sample MS7 at different nitric acid contents. (a) 3.34, (b) 2.0, (c) 1.0, (d) 0.5, and (e) 0.2 mol relative to 100 mol water. Image (a) contains the corresponding TEM image. Figure 5 SEM images of sample MS12 at different sulfuric acid contents. (a) 1.0 and (b) 2.0 mol relative Vorinostat to 100 mol water. No growth was observed with the 3.34 molar ratio. Microstructural properties studied by XRD and N2

sorption isotherms were collectively presented for all samples in Figure 6 (sorption isotherms) and Figure 7 (XRD patterns) to clarify differences associated Selleckchem MK-2206 with each condition. These data were used to calculate the pore structural properties presented in Table 2. First, we will talk about the sample prepared at 3.34 NA which is the mutual counterpart of the silica fiber sample prepared using HCl; we will then discuss the effect of varying the acid content for both nitric and sulfuric acids. Figure 6 Nitrogen adsorption-desorption isotherms of

mesoporous silica prepared under SB-3CT quiescent interfacial growth method. (a) All samples and (b) samples MS7 and MS12 prepared using various molar ratios of nitric acid (NA) and sulfuric acid (SA), respectively. Some isotherms were shifted upwards for proper comparison. Figure 7 XRD patterns of mesoporous silica products. (a) Samples MS7 and MS12 prepared at different molar ratios of nitric acid (NA) and sulfuric acid (SA) respectively and (b) all remaining samples. Sample MS12 at 3.34 SA is not shown because no product was grown throughout the growth period. As shown in Figure 6a, the sorption isotherms of the spherical silica precipitated at 3.34 NA M are very comparable to those of the fibers. The isotherms have type IV mesoporous isotherms showing capillary condensation step at p/po ~ 0.3 that is absent of any hysteresis. The relatively steep capillary condensation indicates a uniform size distribution with a pore diameter of 2.86 nm (compared to 2.35 nm of MSF) and respective surface area and pore size of 887 m2/g and 0.54 m3/g. The fibers and spherical particles possess comparable pore area properties except that the nitric acid causes a little swelling to the pore size. The pore order of the 3.34 NA sample is reflected in the XRD pattern in Figure 7a.

The suspensions obtained from each soil samples were seeded onto nutritive plates, and incubated in triplicate over a range of temperatures (4, 10, 15 and 22°C). After 30–90 days of incubation, approximately 30 to 60 yeast-like colonies developed on each plate. In contrast, no colonies or low colony numbers (4 to 8) appeared on plates from water samples. Because large numbers of

isolates were obtained, isolates were grouped according to their isolation growth temperature and colony characteristics such as pigmentation, texture, elevation and size. Among the 64 groups, several differed only by isolation growth temperature. These isolates were Pembrolizumab order grown at different temperatures and re-grouped according to macromorphological characteristics at their optimal growth temperature. In this way, 35 groups were ultimately generated. Several isolates from each group (at least one isolate per sampling site; a total of 78 isolates) were selected for molecular and biochemical analyses. Molecular identification of yeasts The chromosomal DNA was purified from cultures of each yeast isolate and the D1/D2 region of 26S rDNA and the ITS1-5.8S- ITS2 (hereafter designated the ITS region for simplicity) regions of the rDNA were amplified Palbociclib order by PCR. The amplicons obtained were purified from gels and sequenced on both strands. Isolates showing 100% identity in both rDNA sequences

were grouped and their DNA sequences were submitted to GenBank under the accession numbers listed in Table 1. Species identification was performed

by comparison with the GenBank references, using as criterion the Blast-hits with ≤ 0.5% difference with the query [14]. In 84% of the isolates the closest Blast-hits obtained for both rDNA sequences were coincident. When this PAK5 was not the case, the D1/D2 results were used for identification because they yielded higher identity percentages than did the ITS (see Additional file 1). 76% of the isolates could be identified to species level by this molecular analysis. 22 species belonging to12 genera were identified, of which 80 and 20% were Basidiomycetes and Ascomycetes, respectively. The genera containing the highest number of species were Mrakia (5 species) and Cryptococcus (4 species). However, the species Sporidiobolus salmonicolor was the most abundant, being identified in 24 isolates from 13 different sampling sites. Mrakia gelida was the only yeast species present in both water and soil samples. Of the three isolates identified as Leuconeurospora sp., two of them (T11Cd2 and T27Cd2) possessed identical D1/D2 and ITS sequences, both of which differed from the third (T17Cd1) by 0.7%. However, the macromorphological characteristics of the three isolates, including pigmentation, differed markedly under identical culture conditions (see Additional file 2). Because of these discrepancies, the molecular and morphological analyses were repeated several times, but the results were highly consistent.

RNA samples of the four RG7204 manufacturer biological replicates were reverse-transcribed and labeled according to the protocols detailed in http://​www2.​surrey.​ac.​uk/​fhms/​microarrays/​Downloads/​Protocols/​. For each time-point and strain the cDNA samples from two biological replicates were labeled with Cy3 and two with Cy5. Each mutant cDNA sample was cohybridised with the corresponding (matched timepoints and opposite dye orientation) wild-type cDNA to arrays according to a ‘Balanced Block Design’ [27], as outlined in Figure  1. In addition, direct comparisons of M145 48 h vs M145 18 h and M145 36 h vs M145 18 h cDNA were conducted, also with a balanced block design, to reveal genes changing during

MG-132 research buy normal development of the wild type. Thus, a total of 32 arrays were used in this analysis. After scanning with an Affymetrix 428 array scanner, the images were processed with BlueFuse 3.1 software (BlueGnome). Array data were analyzed using R [54] and the Bioconductor [55] package limma [56, 57]. Raw data were transformed to log2 scale and normalized by applying print-tip loess to each array followed by an across array normalisation (‘scale’ function in the limma package). Because equal dyes are needed in the balanced block design, only genes having at least one good spot on all four arrays of a particular comparison were considered in further analysis. Differential significance between conditions was determined by

using the eBayes function of limma; resultant p-values were corrected by the application of Benjamini and Hochberg “false discovery rate” correction [28]. A difference in gene expression was considered significant if it had an adjusted p-value <0.05. The microarray data have been deposited with ArrayExpress (Accession number E-MTAB-1942). Quantitative real time PCR (qRT-PCR) RNA samples, isolated as described above, were further treated with RQ1 RNase-free DNase (Promega) to remove all traces of DNA. DyNAmo™ SYBR® Green 2-Step qRT-PCR kit (Finnzymes) was used to generate cDNA and reactions were carried out at 45°C Sulfite dehydrogenase for 1 h using 15 ng of random hexamers

primers and 1 μg of total RNA. Two biological replicates of the RNA were used and three independent qRT-PCR reactions were run for each of them, i.e. six in total for each strain and time point. Quantitative real-time PCR of selected genes was performed using a Rotor-Gene 2000 Real-time cycler (Corbett Research). Two μl of a 1:5 dilution (in 10 mM Tris–HCl pH 8.0) of first strand cDNA reaction was used as a DNA template in a 20 μl final reaction volume of the qPCR using a specific primer pair for each tested gene (Additional file 3: Table S2). hrdB is a constitutively expressed gene encoding the principal RNA polymerase factor of S. coelicolor, and was used as a control for the qRT-PCR experiment. Negative controls with 10 mM Tris–HCl pH 8.0 instead of template were included.