Rok regulates yuaB expression during architecturally complex colony development of Bacillus subtilis 168

Abstract

Transcriptome analysis of a Bacillus subtilis rok strain that showed reduced complex colony structure formation revealed significant downregulation of the yuaB gene. Overexpression of yuaB restored structure formation in the rok strain. We show that transcription of yuaB is indirectly regulated by Rok, independently from its previously described AbrB-dependent regulation.

FIG. 1.

B. subtilis complex colony structure depends on Rok-regulated yuaB transcription. Strains were spotted on 2× SG plates and incubated for 3 days. The scale bar in the lower right corner represents 10 mm. (A) Colony morphology of various B. subtilis strains. (B) B. subtilis yuaB and rok mutants transformed with pDRyuaB1 (P_spank-yuaB) and pDRyuaB2 (P_hyperspank-yuaB) constructs in the absence (upper panels) or presence (lower panels) of 1 mM IPTG.

FIG. 2.

Expression of yuaB is altered in B. subtilis by Rok, AbrB, DegU, SinI, and EPS. Strains were spotted on 2× SG plates and incubated for 3 days. Fluorescence was detected in complex colonies using an Olympus MVX10 macrozoom fluorescence microscope equipped with a PreciseExcite LED fluorescence illuminator (470 nm), a GFP filter set (excitation at 460/480 nm and emission at 495/540 nm), and an Olympus XM10 monochrome camera. For the single-cell analysis of complex colonies, a colony was scraped from the plate using a scalpel and homogenized for 1 min using a mini-beadbeater-8 (Biospec, Bartlesville, OK), and fluorescence was measured using a Coulter Epics XL-MCL flow cytometer (Beckman Coulter, Mijdrecht, Netherlands) operating an argon laser (488 nm). For each sample, at least 20,000 cells were analyzed using WinMDI 2.8 (http://facs.scripps.edu/software.html). The numbers of cells are indicated on the y axis of the graphs, and their relative fluorescence levels are indicated on the x axis on a logarithmic scale. “Gmean” indicates the mean fluorescence of P_yuaB-GFP-containing cells at the single-cell level. The scale bar in the lower left corner represents 10 mm.