Bacterial strains, media and growth conditions
The experiments were conducted with Bacillus subtilis NCIB3610 obtained from Bacillus genetic stock center (Ohio State University, Columbus OH). Frozen glycerol (15%) stocks were revived overnight at 37°C on a rotary shaker in 50 mL Luria-Bertani (LB) broth, containing 10 g L-1 tryptone, 5 g L-1 yeast extract, 5 g L-1 NaCl. For experiments, 1 mL of the overnight culture was freshly inoculated into 50 mL LB and cells were harvested in the mid-exponential phase after ~4-5 h of growth. LB medium fortified with 0.7% agar was used in swarm expansion assays. Minimal salt glycerol glutamate (MSgg) medium was prepared according to Branda et al. [12] containing 5 mM potassium phosphate (pH 7), 100 mM MOPS (pH 7), 0.5% glycerol, 0.5% glutamate, 50 mg L-1 tryptophan, 50 mg L-1 phenylalanine, 2 mM MgCl2, 0.7 mM CaCl2, 50 μM MnCl2, 50 μM FeCl3, 1 μM ZnCl2, 2 μM thiamine. Except for the swarming assay, experiments with 3610Δnos were performed in media supplemented with 1 mg L-1 erythromycin and 25 mg L-1 lyncomycin.
The influence of NO on wild-type B. subtilis was tested with supplementation of NOS inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), NO scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO), and the NO donors S-nitroso-N-acetylpenicillamine (SNAP) for more short-term NO effects (t½ ≈ 50 min; dispersal experiment) or 3,3-Bis(aminoethyl)-1-1-hydroxy-2-oxo-1-triazene (Noc-18) in longer experiments (t½ ≈ 3400 min; swarming and biofilm formation experiments). The 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 nosknock-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 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 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 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. The maximum fluorescence values of single cells were measured and corrected for the cell ambient background.
Biofilm formation
The influence of NOS-derived NO on biofilm formation was tested by investigating the morphology and fine structure of spot colonies grown on MSgg fortified with 1.5% agar. Additionally, the amount of vegetative cells and spores in biofilms grown on the liquid-air interface ('pellicles') in MSgg medium was quantified. Both agar and medium were supplemented with sterile filtered (0.2 μm, Spartan, Millipore, Schwalbach, Germany) 100 μM L-NAME, 75 μM c-PTIO or 130 μM Noc-18 after autoclavation.
Colony morphology was investigated in 6-well microtiter plates (Nunclon Surface, Nunc, Denmark) and colony fine structure was investigated in Petri dishes (Sarstedt, Nümbrecht, Germany). The wells of the microtiter plates were filled with 6 mL and the Petri dishes with 25 mL MSgg agar. After the agar dried for ~ 16 h at room temperature (RT), 5 μL of a LB-grown overnight culture was spotted on the agar surface, dried open for 10 min in a laminar flow hood, and incubated at 26°C. Fine structure of 3 days old colonies was visualized by illuminating the sample with an external light source (swan neck lamp, KL 1500 electronic, Schott, Mainz, Germany) and capturing reflected light with a DS-Q1-MC CCD camera (Nikon, Japan) mounted on a light microscope (DM RA2, Leica, Solms, Germany) equipped with Leica 5× NA0.15 HC PL Fluotar lens. Whole colony morphology was documented with a digital camera after 4 days of growth.
Pellicle formation was quantified in glass test tubes containing 25 mL MSgg medium. MSgg tubes were inoculated with 25 μL of mid-exponential phase culture and incubated for 7 days at 26°C without agitation. Directly after the inoculation 980 μL medium was removed from the tube and subjected to NO staining with CuFL as described above. During the course of biofilm formation 3 vials of each treatment per day were sacrificed for determination of viable cell and spore counts. Biofilms were homogenized in the MSgg medium by sonication (Labsonic U, B. Braun, Melsungen, Germany) for 10 min at ~ 40 W on ice. The cells were plated on LB agar, and incubated 24 h at 26°C to determine the number of colony forming units (cfu). Spore counts were determined from the same samples by subjecting a part of the homogenates to pasteurization for 20 min at 80°C in a water bath prior to plating. O2 and NO concentrations in the biofilm incubations were measured with microsensors as previously described [43, 44].
Swarm expansion assay
Swarm experiments were conducted as described by Kearns and Losick [13]. Briefly, cells grown in LB at 37°C to the mid-exponential phase were harvested by centrifugation (15 min, 4000 RCF, 15°C) and re-suspended in phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4) containing 0.5% ink. Swarm plates were prepared in Petri dishes (diameter = 8.5 cm) by pouring 25 mL LB fortified with 0.7% agar and supplemented with 100 μM L-NAME, 100 μM c-PTIO or 20 μM and 200 μM Noc-18. The plates were dried for 30 min under a laminar flow hood, directly afterward inoculated with 3 × 108 cells within 10 μL in the centre of the plate, dried for another 10 min, and incubated at 37°C. The swarm radii were measured relative to the origin of swarming, which was demarked by the edge of the ink stained agar in the centre.
We used statistics to confirm that the differences between treatments were not significant. Normality of the data was confirmed with Saphiro-Wilk W test (α = 0.01). Comparison between different experimental treatments was performed by a One-Way-Analysis of Variance (α = 0.01) with NCSS software (PASS2000, Kaysville, UT). Turkey-Kramer post-hoc test was used to determine significant differences between individual factors.
Dispersal assay
Spot colony biofilms were grown on agar in 6-well plates filled with MSgg agar, MSgg agar + 100 μM L-NAME and MSgg agar + 75 μM c-PTIO. After 4 days of growth a 100 μL drop MSgg medium was mounted on the colonies and incubated for 2 h at RT. The drops of the experimental treatments contained 100 μM L-NAME for MSgg/L-NAME agar, 750 μM c-PTIO for MSgg/c-PTIO agar, 300 μM SNAP for MSgg agar, and 100 μM L-NAME + 300 μM SNAP for MSgg/L-NAME agar. Next, 80 μL of the drop liquid were removed. The cells were fixed with formaldehyde at a final concentration of 3.7% and incubated at 4°C overnight. Cell counting was done with a flow cytometer (FACS Calibur, Becton Dickinson, Franklin Lakes, NJ) on the following day. The fixed cells were mixed with 500 μL sterile filtered, deionised water that contained fluorescent latex beads (AlignFlow, alignment beads 2.5 μm, Molecular Probes, Eugene, OR) and with 1×Cybr Green DNA stain (Molecular Probes, Eugene, OR). Vegetative cells were differentiated from spores based on their size difference. Cell counts per volume could be calculated based on the number of beads counted in each run and an initial calibration of the bead solution.
Germination assay
MSgg medium was supplemented with the same treatments as used during the dispersal assay. Spores were prepared by growing B. subtilis in Difco sporulation medium (DSM) at 37°C for 16 h. After that time all cells in DSM were spores as determined by comparing direct plate counts to heat inactivated (80°C, 20 min) plate counts. Spores were added to MSgg and MSgg plus treatments to reach a final concentration of ~106 spores mL-1. 100 μL drops of the MSgg-spore suspensions were placed on sterile Petri dish surfaces and incubated for 2 h at RT. 80 μL of each drop were harvested and split in two parts: 40 μL were plated immediately on LB agar to determine the total cfu (vegetative cells + spores), while the other 40 μL were heated at 80°C for 20 min prior to LB-plating to determine the spore forming units.
Microsensor measurements
NO microprofiles were measured in the same set-up as used in the dispersal assay. Spot colony biofilms were grown on MSgg agar in Petri dishes for 4 d. A 100 μL drop of MSgg was mounted on top of the biofilm and NO microprofiles were measured immediately with an NO microsensor as described previously [43]. For each experimental treatment, MSgg was supplied either with or without 300 μM of the NO donor SNAP. SNAP was mixed to MSgg directly before the experiment. Experimental treatments were as followed: (i) wild-type: B. subtilis 3610 for which MSgg agar and drop were added without further supplementation; (ii) wild-type: B. subtilis 3610 for which MSgg agar and drop were supplemented with 100 μM L-NAME; and (iii) B. subtilis 3610 Δnos for which MSgg agar and drop were added without further supplementation.