FacX is resistant to boiling, but sensitive to Proteinase K.

<p>(A) Conditioned media was boiled for 15 min and utilized in the sporulation assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144168#pone.0144168.g002" target="_blank">Fig 2A</a>) with strain MF1913 (P_{<i>hy-spank</i>}-<i>kinA</i>). (B) Conditioned media was treated with proteinase K and utilized in the sporulation assay with strain MF1913 (P_{<i>hy-spank</i>}-<i>kinA</i>). When indicated, KinA was induced with IPTG (20 μM). Cells were grown for 2 hrs at 37°C before image capture. Membranes were stained with TMA.</p

KinA-dependent sporulation requires a threshold level of FacX.

<p>MF1913 (P_{<i>hy-spank</i>}-<i>kinA</i>) was grown to exponential phase and 0.2 ml of culture was used to inoculate either fresh CH spiked with 37.5 ul of 67X concentrated conditioned media (1X final concentration)(top) or diluted conditioned media (3 parts conditioned CH to 1 part fresh CH) (bottom). When indicated, <i>kinA</i> expression was induced with IPTG (20 μM). Cells were grown for 2 hrs (top panels) or 2.5 hrs (bottom panels) at 37°C before image capture. Membranes were stained with TMA.</p

FacX activity is not dependent on Spo0A, SigH, or ComX for production, and does not require the Opp or App peptide uptake systems.

<p>(A) A strain harboring P_{<i>hy-spank</i>}-<i>kinA</i> (MF1913) was grown 2 hrs in the indicated media at 37°C before image capture. (B) A strain containing P_{<i>hy-spank</i>}-<i>kinA</i> in an Δ<i>oppABCDF</i>, Δ<i>appA</i> double mutant (BQA121) was grown 2 hrs at 37°C before image capture. The conditioned media utilized in this experiment was obtained from PY79. When indicated, KinA expression was induced with IPTG (20 μM). Membranes were stained with TMA.</p

<i>Bacillus</i> can be induced to sporulate through expression of a constitutively active allele of Spo0A (Spo0A*) if induced during stationary phase or grown in conditioned media.

<p>(A) A strain harboring P_hy-spank—<i>Spo0A*</i> (MF2146) was grown in CH at 37°C, induced by the addition of IPTG (200 μM) at the indicated optical densities, and grown for 2 hrs at 37°C before image capture. Membranes (white and red) were stained with FM4-64, and DNA (green) was stained with DAPI. (B) A strain harboring P_{<i>hy-spank</i>}-<i>Spo0A*</i> (MF2146) was grown in conditioned media obtained from PY79 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144168#pone.0144168.g002" target="_blank">Fig 2A</a>. When indicated, Spo0A* was induced by the addition of IPTG (either 200 μM or 500 μM, as indicated, beginning OD₆₀₀ 0.04) and grown for 2 hrs at 37°C before image capture. Membranes were stained with TMA.</p

FacX activity is dialyzable.

<p>Conditioned CH medium was dialyzed against fresh CH using dialysis tubing with the indicated molecular weight cutoff. The dialysate was then used to perform the KinA-dependent sporulation assay as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144168#pone.0144168.g002" target="_blank">Fig 2A</a> with strain MF1913. When indicated, <i>kinA</i> expression was induced with IPTG (20 μM). Cells were grown for 2 hrs at 37°C before image capture. Membranes were stained with TMA.</p

Media from post-exponential growth contains a protein-based factor that promotes sporulation via KinA induction.

<p>(A) A typical PY79 growth curve in CH medium at 37°. The arrow indicates region in the growth curve (OD₆₀₀ between 1.3 and 1.5) where the conditioned media (CM) was collected. The shaded region indicates the region of the growth curve in which exponential growth occurs. (B) Schematic representation of the KinA-dependent sporulation assay used throughout this study. (C) A strain harboring P_{<i>hy-spank</i>}-<i>kinA</i> (MF1913) was grown as described in (A) with conditioned media collected from PY79. KinA was induced with IPTG (20 μM final concentration). Membranes were stained with TMA.</p