The Combretastatin A4 mw theoretically expected time courses of NO release by the donors without concurrent loss processes in different experiments are shown in the additional file 1 (figures

s1 and s2). Construction of nos knock-out Deletion of nos gene from B.subtilis PY79 genome was achieved by long-flanking homology polymerase chain reaction (LFH-PCR) technique [37]. The deletion/insertion nos::mls was constructed by PCR amplifying approximately 1 kbp from 5′-flanking region of nos gene with primers P1b_BsNOS (5′ taa cgg cat aca aca ttc cgg agg 3′) and P2b_BsNOS (5′ att atg tct ttt gcg cag tcg gcc ttt ttc ttc caa caa act ctc ccc 3′), while another band of near 1 kbp from 3′-flanking region was amplified using P3_BsNOS (5′ cat tca att ttg agg gtt gcc agc aat cgt taa gcc gaa cta ttt tta tc 3′) and P4_BsNOS (5′ cgc gaa ctg gac gga tat gcc tt 3′). The resulting PCR products were then used as primers to amplify the erythromycin-resistance cassette from the plasmid pDG646 [38] as previously selleck compound described [37]. This creates a deletion of the nos gene from nucleotide +12 to +1064 assuming the +1 nucleotide described in Adak et al. [5]. The PCR products were then transformed into PY79 as previously described

[39] and the mutants were confirmed by PCR. The nos::mls mutation were then introduced in 3610 strain by SPP1 phage transduction [40, 41] and confirmed by PCR analysis. Detection of intracellular NO formation One milliliter overnight culture was inoculated in 50 mL LB and in 50 mL LB supplemented with 100 μM NOS inhibitor L-NAME. The culture was grown to the mid-exponential phase and was mixed with the NO sensitive dye CuFL (prepared according to suppliers instruction; Strem Chemicals, Newburyport, MA) [42] to reach a final selleck concentration of 10 μM. In addition, cells grown to the mid-exponential phase in LB without L-NAME were mixed with NO scavenger c-PTIO to a final concentration of 100 μM and incubated for 1.5 h at room temperature prior to CuFL staining. Cells were incubated with CuFL for ~30 min, placed on microscopic glass slides and covered

with poly-L-Lysine coated cover slips. NO imaging was performed PAK5 with a Confocal Laser Scanning Microscope (LSM 510, Zeiss, Germany) equipped with a Plan-Apochromat 100×, NA 1.4 oil lens. CuFL was excited at a wavelength of 488 nm with an Argon ion laser. The beamsplitter in front of the laser was HFT 488/543. The detector was equipped with a bandpass filter BP 505-530. In a second scanning cycle transmission images were collected at a wavelength of 633 nm with the in-built photo-diode detector. Digital image processing was done with ImageJ software (National Institute of Health, Bethesda, MD). For quantification of relative fluorescence (representing NO concentrations) images were filtered by a 2 pixel wide gaussian kernel.