Rho inactivation negatively affects biofilm formation in <i>B</i>. <i>subtilis</i>.

<p>(A) Colony (left column) and pellicle (right column) biofilm formation by <i>B</i>. <i>subtilis</i> NCIB 3610 WT and RM strains. Relevant genotypes are indicated on the side of each image. The colony column shows individual colonies grown on MSgg agar medium for 72h at 30°C. The scale bar is 5mm. The pellicle column shows microtitre wells (diameter 1.5 cm) in which cells were grown in MSgg medium without agitation for 72h at 30°C. The scale bar is 5 mm. For the colony assay, 2μl of culture was spotted onto MSgg agar plate. For pellicle assay, 2μl of culture was added to 2ml of MSgg medium in a well of 24-well sterile microtiter plate. The experiment was reproduced at least five times including four replicas for each strain. Presented are the results from the representative experiment. (B and C) Rho inactivation decreases expression of biofilm-specific genes. Promoters of the operons <i>epsA-O</i> (B) and <i>tapA-sipW-tasA</i> (C) encoding exopolysaccharides and amyloid-like protein fibers, respectively, were fused to the butterfly luciferase gene <i>luc</i> and tested for transcription activity in <i>B</i>. <i>subtilis</i> BSB1 WT (blue lines) and RM (red lines) cells grown in liquid biofilm-stimulating medium MSgg as described in Materials and Methods. The thin double lines depict growth curves measured by optical density OD 600nm and the thick lines show relative luminescence readings corrected for OD. The unmarked individual points of the curves are the means of the values measured at the 5 min intervals in four independent isogenic cultures during the same experiment. The experiment was reproduced three times and the results of the representative one are shown. (D) <i>B</i>. <i>subtilis</i> genomic region corresponding to the <i>epsA-O</i> operon shows asRNA of ~15740 nt long starting from the <i>epsO</i> gene and overlapping the entire operon in 1012 RM strain. The double slash marks represent internal part of <i>eps</i> operon (<i>epsE-epsF-epsH-epsI-epsJ-epsK-epsL-epsM-epsN</i>) that is not shown (the whole operon can be visualized on <a href="http://genome.jouy.inra.fr/cgi-bin/seb/index.py" target="_blank">http://genome.jouy.inra.fr/cgi-bin/seb/index.py</a>). Sections show annotated genome (top) and expression profiles of WT (black) and RM (red) on the (+) and (–) strands (mid and bottom sections). (E) Schematic illustration of the construction of NCIB 3610 <i>epsO</i>:Ter insertion mutant. Bars tipped with open circles and red dotted arrow denote Rho-independent terminators and <i>eps</i> asRNA respectively; elements not to scale. (F) RT-PCR analysis of antisense transcription of <i>eps</i> operon region (top section) in the NCIB 3610 WT and RM strains. RT-PCR analysis of rRNA (bottom section) was used as a control. Synthesis of cDNA was performed using 50 ng of total RNA as template (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006909#sec019" target="_blank">Materials and methods</a>). Reactions were performed with (+) and without (-) reverse transcriptase (RT). PCR was done with oligonucleotides specific for <i>eps</i> asRNA within <i>epsK</i> gene (asRNA <i>eps</i>) and for rRNA (rRNA) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006909#pgen.1006909.s016" target="_blank">S7 Table</a>).