Identification of genes required for different stages of dendritic swarming in Bacillus subtilis, with a novel role for phrC

Abstract

Highly branched dendritic swarming of B. subtilis on synthetic B-medium involves a developmental-like process that is absolutely dependent on flagella and surfactin secretion. In order to identify new swarming genes, we targeted the two-component ComPA signalling pathway and associated global regulators. In liquid cultures, the histidine kinase ComP, and the response regulator ComA, respond to secreted pheromones ComX and CSF (encoded by phrC) in order to control production of surfactin synthases and ComS (competence regulator). In this study, for what is believed to be the first time, we established that distinct early stages of dendritic swarming can be clearly defined, and that they are amenable to genetic analysis. In a mutational analysis producing several mutants with distinctive phenotypes, we were able to assign the genes sfp (activation of surfactin synthases), comA, abrB and codY (global regulators), hag (flagellin), mecA and yvzB (hag-like), and swrB (motility), to the different swarming stages. Surprisingly, mutations in genes comPX, comQ, comS, rapC and oppD, which are normally indispensable for import of CSF, had only modest effects, if any, on swarming and surfactin production. Therefore, during dendritic swarming, surfactin synthesis is apparently subject to novel regulation that is largely independent of the ComXP pathway; we discuss possible alternative mechanisms for driving srfABCD transcription. We showed that the phrC mutant, largely independent of any effect on surfactin production, was also, nevertheless, blocked early in swarming, forming stunted dendrites, with abnormal dendrite initiation morphology. In a mixed swarm co-inoculated with phrC sfp + and phrC + sfp (GFP), an apparently normal swarm was produced. In fact, while initiation of all dendrites was of the abnormal phrC type, these were predominantly populated by sfp cells, which migrated faster than the phrC cells. This and other results indicated a specific migration defect in the phrC mutant that could not be trans-complemented by CSF in a mixed swarm. CSF is the C-terminal pentapeptide of the surface-exposed PhrC pre-peptide and we propose that the residual PhrC 35 aa residue peptide anchored in the exterior of the cytoplasmic membrane has an apparently novel extracellular role in swarming.

Models of Self-Organizing Bacterial Communities and Comparisons with Experimental Observations

“…Recent studies [8], using genetic analysis and fluorescent microscopy to measure the level of production (expression) from the gene encoding the major flagellum subunit in situ, have identified a specific subpopulation of hyper-flagellated cells ('swarmers' ). These are dominant in the formation of buds and then subsequently spearhead dendrites in the tips.…”

Laboratory Strains ofBacillus subtilisDo Not Exhibit Swarming Motility

“…Some labs have tested swarming motility on LB medium in which tryptone was replaced by an equal amount of peptone (13). We reproduced the "LB" medium containing peptone and found that whereas strain 3610 was swarming proficient, strain 168 was swarming deficient (Fig.…”

“…In some cases of the reported swarming of strain 168, plates were poured 1 h before use, dried for 5 min, and incubated at 60 to 70% humidity (13). When 0.7% agar LB plates were freshly poured and not dried, we noticed that toothpick inoculation of the cells disturbed the agar surface and caused a pool of water to well forth from the agar (see Fig.…”

New Tools for Comparing Microscopy Images: Quantitative Analysis of Cell Types in Bacillus subtilis

“…For those experiments, alternative methods, such as flow cytometry or colony thin sectioning, are used (27,28). In some cases, macroscopic colonies can still be subjected to microscopy, but only when images are taken at the colony edge, where there is a monolayer of cells, or when the colonies are dissected before microscopy (28)(29)(30)(31)(32)(33). Studies that perform microscopy on macroscopic colonies typically show qualitative results, such as representative images with fluorescent overlays (e.g., see the work of Fall et al [29] and López et al [32]).…”