Estimation of the State of the Bacterial Cell Wall by Fluorescent In Situ Hybridization

Abstract

Fluorescent in situ hybridization (FISH) is now a widely used method for identification of bacteria at the single-cell level. With gram-positive bacteria, the thick peptidoglycan layer of a cell wall presents a barrier for entry of horseradish peroxidase (HRP)-labeled probes. Therefore, such probes do not give any signal in FISH unless cells are first treated with enzymes which hydrolyze the peptidoglycan. We explored this feature of FISH to detect cells which have undergone permeabilization due to expression of autolytic enzymes. Our results indicate that FISH performed with HRP-labeled probes provides a sensitive method to estimate the states of cell walls of individual gram-positive bacteria.

Knockout of the alanine racemase gene inLactobacillus plantarumresults in septation defects and cell wall perforation

“…In order to explain the strong reduction of viability of the MD007 mutant during D D -Ala starvation without optical density decrease, the integrity of the cell envelope was assessed by two different fluorescent techniques and by monitoring the release of a cytoplasmic marker (lactate dehydrogenase). The fluorescent in situ hybridization (FISH) technique was first used to monitor cell wall permeability, as this approach has been successfully implemented for similar purposes with other lactic acid bacteria [25]. The EUB338-Cy3 molecular probe (20-mer oligonucleotide linked to Cy3, targeted to the 16S rRNA, $6 kDa) was hybridized with the wild-type and mutant cells.…”

Natural whey starter for Parmigiano Reggiano: culture-independent approach

“…Specimens were treated by covering the spots with 10 ll of proteinase K (10 mg ml )1 ) at 37°C for 10 min, to allow permeabilization of Grampositive cells. Lbh1-fluorescein iso-thiocyanate (FITC)labelled probe (Bidnenko et al 1998), specific for Lact. helveticus, and St4-Cy3-labelled probe specific for Streptococcus thermophilus (Mercier et al 2000) have been used.…”

Cell Walls and the Convergent Evolution of the Viral Envelope

“…We calculated the volume of the protein (V) and used this to calculate its diameter. The average density of a protein of 60 kDa can be calculated as described previously (100,101), resulting in 1.4114 g/cm 3 . The volume for a protein of this size is then calculated as follows: We declare that we have no conflicts of interest.…”

“…The cell wall is also a selective filter, allowing free diffusion of small molecules and ions. Experiments with cell walls in plants and bacteria have determined an exclusion size of approximately 50 to 60 kDa (3)(4)(5). This allows the diffusion of important signaling molecules, such as phytohormones in plants, but not virus particles.…”

“…ϭ ͕[1 ⁄ p(g ⁄ cm3 ) ϫ 10 21 (nm 3 ⁄ cm3 )] ⁄ Na(Da ⁄ g)͖ ϫ M(Da)(1) V(nm 3 ) ϭ ͕[0.70851(cm 3 ⁄ g) ϫ 10 21 (nm 3 ⁄ cm 3 )] ⁄ Na(Da ⁄ g)͖ ϫ M(Da) (2) V(nm 3 ) ϭ [7.08516(nm 3 /g) ⁄ Na(Da ⁄ g)] ϫ M(Da) (3) V(nm 3 ) ϭ 0.00117 (nm 3 /Da) ϫ M(Da) (4) V(nm 3 ) ϭ 0.00117(nm 3 /Da) ϫ 60, 000(Da) (5) V ϭ 70.579(nm 3 ) (6)where V is the volume of the protein, p is the density of the protein (in grams/cubic centimeter), M is the mass of the protein (in daltons), and Na is Avogadro constant.Assuming a sphere with volume V, the diameter (d) is calculated as follows:d(nm) ϭ 2 ϫ (3V ⁄ 4 1⁄3 )(7)d(nm) ϭ 2 ϫ [3 ϫ 70.579(nm 3 ) ⁄ 4 1⁄3 ] This work was supported by a National Health and Medical Research (NHMRC) Australia Fellowship awarded to E.C.H.…”