Incubation of wild-type cells in LB with the NO 3-deazaneplanocin A clinical trial synthase (NOS) inhibitor L-NAME and of a mutant that lacked the nos gene decreased in both cases NO production to ~ 7% as compared to untreated wild-type cells (Figure 1C-E). In contrast, supplementing MSgg medium with the NOS inhibitor L-NAME and growing the nos mutant

EGFR inhibitor in MSgg decreased NO production to only 85% and 80%, respectively, as compared to untreated wild-type cells (Figure 1E). Figure 1 Nitric-oxide-synthase (NOS)- derived NO formation by B. subtilis 3610. (A-D) Confocal laser scanning micrographs of cells grown in LB for 4 h at 37°C. Shown is the overlay of: gray – transmission and green – fluorescence of NO sensitive dye CuFL. (A) Wild-type without supplements, (B) supplemented with 100 μM c-PTIO (NO scavenger), (C) 100 μM L-NAME (NOS inhibitor), and (D) 3610Δnos. Scale bar is 5 μm. (E) Single-cell quantification of intracellular NO formation of cells grown in LB (gray bars) MLN2238 and MSgg (white bars) using CuFL fluorescence intensity

(A.F.U. = Arbitrary Fluorescence Units). Error bars show standard error (N = 5). The data shows that B. subtilis uses NOS to produce NO in LB and indicates that NO production via NOS is low in MSgg. Furthermore, the NO scavenger c-PTIO effectively reduces intracellular NO and the NOS inhibitor L-NAME inhibits NO formation by NOS. Hence, both compounds are suitable tools to test the effect of NO and NOS-derived NO, respectively, on multicellular traits of B. subtilis. Moreover, the data indicates that B. subtilis produces significant amounts of NO with an alternative mechanism besides NOS when grown in MSgg. An alternative pathway of NO formation in B. subtilis could

be Ponatinib in vivo the formation of NO as a by-product during the reduction of NO2 – to ammonium (NH4 +) by the NO2 – reductase NasDE [25]. Both LB (~35 μM) and MSgg (~ 5 μM) contained traces of oxidized inorganic nitrogen (NO3 – or NO2 -; NOx), which might be a sufficient source for low nanomolar concentrations of NO even if most NOx is reduced to NH4 +. Gusarov et al. [26] showed that NasDE actively reduces NOx in LB-cultures at the end of the stationary phase. However, NO production from ammonifying NO2 – reductases has so far only been reported for the ammonifying NO2 – -reductase Nrf of E. coli [27], but not for NasDE of B. subtilis. The potential ability of NasDE to generate NO may be an interesting subject for further research directed toward the understanding of how B. subtilis controls NO homeostasis under different environmental conditions. NO is not involved in biofilm formation of B. subtilis 3610 We tested the influence of NOS-derived NO and exogenously supplemented NO on biofilm formation of B. subtilis 3610 by monitoring the morphology of agar-grown colonies and the development of biofilms on the air-liquid interface (pellicles) in MSgg medium.