The extracellular matrix protein TasA is a developmental cue that maintains a motile subpopulation within Bacillus subtilis biofilms
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PROVIDER: PRJNA574155 | ENA |
REPOSITORIES: ENA
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Project description:In nature, bacteria form biofilms – differentiated multicellular communities attached to surfaces. In the otherwise sessile biofilms, a subset of cells continues to express motility genes. Here, we demonstrate a biological role for this subpopulation, which enabled biofilms of Bacillus subtilis to expand on high-friction surfaces. Moreover, we show that the extracellular matrix (ECM) protein TasA was required for the expression of flagellar genes. In addition to its structural role as an adhesive fiber for cell attachment, TasA served as a developmental signal similar to ECM proteins of multicellular organisms. Transcriptomic analysis revealed that TasA regulated a specific subset of genes, inducing genes involved in motility and repressing ECM production. A screen for suppressor mutations that restore motility in the absence of TasA revealed activation of the biofilm-motility switch by the two-component system CssRS, that alleviated Spo0A repression by TasA. Our results suggest that although mostly sessile, biofilms retain a degree of motility by maintaining a motile subpopulation.
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Project description:The genus Lactobacillus contains over 100 different species that were traditionally considered to be uniformly non-motile. However, at least twelve motile species are known to exist in the L. salivarius clade of this genus. Of these, Lactobacillus rumnis is the only motile species that is also autochthonous to the mammalian gastrointestinal tract. The genomes of two L. ruminis strains, ATCC25644 (human isolate, non-motile) and ATCC27782 (bovine isolate, motile) were sequenced and annotated to identify the genes responsible for flagellum biogenesis and chemotaxis in this species. Transcriptome analysis revealed that motility genes were transcribed at a significantly higher level in motile L. ruminis ATCC27782 than in non-motile ATCC25644 during the motile growth phase. In preparation for RNA isolation from L. ruminis ATCC27782 and ATCC25644 cultures in the motile phase, 20 ml aliquots of MRS broth were inoculated (0.25 % inoculum) with a turbid culture of the desired strain. The cultures were incubated anaerobically at 37 °C for 15 hr. In preparation for RNA isolation from L. ruminis ATCC27782 and ATCC25644 cultures in the non-motile growth phase, 20 ml aliquots of MRS broth were inoculated (1 % inoculum) with a turbid culture of the desired strain. The cultures were incubated anaerobically at 37 °C for 16-20 hrs. RNAprotect Bacteria reagent (Qiagen) was used to stabilize gene expression in the target cultures, and total RNA was isolated according to the protocol for enzymatic and mechanical disruption of bacteria as described in the RNAprotect Bacteria Reagent handbook. RNA isolation was completed with the RNeasy Mini kit, and contaminating DNA was removed with the Turbo DNA-free kit (Ambion). An Oligo aCGH/ChIP-on chip hybridization kit (Agilent) was used for hybridization of the labelled cDNA to the microarrays. Probe hybridization took place at 65 °C for 20 hrs with constant rotation (10 rpm). Microarrays were scanned using the Agilent Microarray Scanner System (G2505B) and the scanned files were converted to data files with Feature Extraction software (Agilent). Three microarrays, (three biological replicates) were used to examine gene transcription during the motile growth phase. One microarray was used to examine gene transcription during the non-motile growth phase.
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