Title

Role of mechanosensitive channels in growth and differentiation of Bacillus subtilis

Date of Completion

January 2008

Keywords

Biology, Microbiology|Chemistry, Biochemistry|Biophysics, General

Degree

Ph.D.

Abstract

This work investigates the role of stretch activated mechanosensitive (MS) channels in growing cells and spores of the bacterium Bacillus subtilis. MS channels are membrane proteins that detect mechanical forces acting on the cell membrane causing the MS channels to open or close. The genome of B. subtilis harbors genes that likely encode MS channels of large (MscL) and small (MscS [YkuT, YhdY, and YfkC]) conductance, the main MS channels. ^ Gene expression studies using translational lacZ fusion showed that MS channels are expressed in growing cells of B. subtilis with expression of mscL being >> mscS genes. The expression of the genes was variably enhanced by changes in the osmolarity of the growth medium. Low mscL-lacZ activity was detected in dormant spores. ^ Strains lacking mscL and/or mscS grew normally in LB medium containing low or high salt. However, log phase cells of strains lacking mscL or mscS and mscL were very sensitive to an osmotic downshock ≥ 0.5 M NaC1 that caused cell lysis, but cells developed osmotic resistance at late log and stationary phase. Strains lacking mscL and one or more mscS genes took longer to develop osmotic resistance, with mscL ykuT and a quadruple mutant (mscL ykuT yhdY yfkC) taking ∼ 3 hr longer than the other strains to develop full osmotic resistance. ^ The MS channel mutants formed normal spores which accumulated optimal levels of osmolytes such as dipicolinic acid, glutamate and arginine as determined by phase contrast microscopy and 1H NMR spectroscopy. The mutant spores germinated normally in presence of various germinants including L-alanine, dodecylamine, and high pressure and also exhibited normal wet heat resistance. ^ Western blot and fluorescence microscopy of GFP-tagged MS channels showed that MscL-GFP is maximally expressed early during log phase while MscS-GFP proteins are maximally expressed in mid to late exponential phase. Subcellular localization of the proteins by Western blotting showed that they are associated with the cell membrane. ^ Overall, this study shows that MS channels in B. subtilis play a critical role in regulation of cell's internal turgor pressure, but are not essential for growth, sporulation or spore germination. ^