B. subtilis - prevalent inhabitant of soil, belonging to aerobic, spore-forming genus Bacillus, establish symbiotic relationship with the plants by forming robust biofilms on the roots surface. Biofilms, prevalent form of existence of microorganisms in every ecosystems, are viewed as highly-structured communities of bacteria embedded in an extracellular matrix. In laboratory conditions the most common form of this structure formed by B. subtilis are pellicles - floating biofilms formed at air-liquid interface. In case of B. subtilis, eps operon employing 15 genes, seems to participate in exopolysaccharide (EPS) synthesis, which is the main component of mentioned matrix. However, the function of particular genes remain unknown. Searching for influence of proteins encoded by those genes on multicellular behaviours, we performed series of insertional mutations in distal part of the eps operon including eight genes (yveS, yveT, yvfA, yvfB, yvfC, yvfD, yvfE and yvfF). Obtained results, with usage crystal violet staining, showed drastic decrease in the amount of produce EPS. Furthermore, mutant strains, excluding the last gene of the eps operon, was unable to develop biofilm community (yveS and yveT mutants) or produced only fragile peaces of pellicles accumulating in the bottom of the cultivation flask. Consequently, the architecture of the colony on a solid agar medium of mutant strains were considerable different from colony formed by parental strain 168. Confocal microscopy (CLSM) as well, confirmed significant changes in three-dimensional structures of those fragments of pellicles in comparison to mature biofilms form by wild strain at the air/liquid interface. The main differences is irregular, chaotic structure due to the lack of bundles, defined as long chains of morphological changed cells. What is interesting, IPTG-dependent induction of pspac promoter driving the expression of every gene in constructed fusions, led to restore biofilm formation ability only in case of yveS and yveT genes.
Relying on coordinated movement, generating by successive waves of moving cells on solid media- swarming is believed to share some features with biofilm formation process. Since, exopolysaccharide was proved to posses properties facilitating active movement in case of Gram-negative bacteria, we decided to check if this is the issue in case of B. subtilis. The first set of experiments showed that, strains harboring mutations in group of yveS-yvfF genes, displayed a completely nonswarming phenotype in 168 background on Luria-Bertani solid medium. However, introduction gene encoded surfactin, crucial for swarming on fully defined media, caused restoration of swarming ability in mutant defective in EPS synthesis. It was found, that the colonizing behaviour and the pellicle formation of Bacillus subtilis depend on producing EPS. This is another step in confirming hypothesis that biofilm and swarming can have overlapping control mechanisms.

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1. Mapping transcription start site of gene encoding protein phosphatase PrpE in Bacillus subtilis
2. Analysis of biofilm formation by natural isolates of Bacillus spp.