Team:LMU-Munich/Data/Knockout

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Latest revision as of 01:56, 27 September 2012

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Knockouts of germination genes

We incubated our mutant strains in Difco sporulation media (DSM) shaking at 37°C for 18-24 hours to induce sporulation. We heated 1 mL of each of the DSM cultures at 80°C for one hour to kill vegetative cells, and used 1 mL at room temperature (allowing vegetative cells to survive). From here, we quantified the cells and spores per mL of each culture using a Neubauer cell counting chamber. Then on LB-agar, we plated different dilutions of the cultures, with heated and unheated cultures on separate plates. The purpose of plating both heated and unheated cultures was to demonstrate the viability of the unheated vegetative cells in each culture. This makes evident that the lack of colonies on heated plates is not due to the inability of the strain's vegetative cells to grow, but from the inability of spores to germinate into vegetative cells capable of growing. For all germination mutants, vegetative cells were able to grow and form colonies. Plates were incubated at 37°C for at least 24 hours. We then quantified the colonies on each plate, and used this to check for cell growth and calculate the spore germination rate for each mutant strain. For details on this germination assay, see Protocols. The germination assays were carried out several times, and yielded similar results each time. The results are shown in Fig. 1.

Fig 1: The germination rates of B. subtilis germination mutants. All germination rates are relative to wild type 168 (W168), our positive control. W168 showed near 100% germination rates. The mutant spo0A::tet is unable to form spores, and should therefore show no germination (our negative control). Our results were consistent with this expectation. Mutants used were: W168: wild-type 168 spo0A::tet: a sporulation-deficient strain B29: cwlD::kan, sleB::mls B30: gerD::cm, sleB::mls B32: cwlJ::spec, cwlD::kan B40: cwlD::kan, sleB::mls, cwlJ::spec B41: cwlD::kan, sleB::mls, gerD::cm B42: cwlD::kan, cwlJ::spec, gerD::cm B43: gerD::cm, sleB::mls, cwlJ::spec B46: cwlD::kan, cwlJ::spec, gerD::cm, sleB::mls B47: gerD::cm, sleB::mls, cwlJ::spec, cwlD::kan

The remaining germination ability in strain B30 in Fig. 1 seems inconsistent with the retained germination ability in strain B42, because B42 contains mutations to only one of the genes knocked out in B30. For example, we would have expected that B41 or B43, which contain both gerD::cm and sleB::mls should retain some germination ability instead. We could find no evidence of a labeling mix-up that could have caused the inconsistency. But the germination of B42 spores occurred in only one of the many germination assays performed on this strain. Hence we cannot rule out an experimental artifact.

The next figure showing the LB-agar plates from the germination assays (see below) demonstrates the inability of our mutant spores to germinate.

In Fig. 2 below, plates from our germination assay illustrate the process of checking for colonies. This colony growth information was used to calculate the number of germinable spores in each culture.

Fig. 2: Plates of germination mutants. Plates contained heated (to kill vegetative cells and leave only spores to germinate) cultures and unheated (where colonies may come from germinated spores and the vegetative cells) cultures. The mutant spo0A::tet is unable to form spores, and should therefore show no germination (our negative control). The mistake of photographing empty plates against a background of spots was realized after plates had been discarded. Presence of colonies is noted below, for clarity. Mutants used were: W168: Wild-type 168; Heated plate: colonies present; Unheated plate: colonies present spo0A::tet: A sporulation-deficient strain; Heated plate: no colonies; Unheated plate: colonies present B42: cwlD::kan, cwlJ::spec, gerD::cm; Heated plate: colonies present; Unheated plate: colonies present B47: gerD::cm, sleB::mls, cwlJ::spec, cwlD::kan; Heated plate: no colonies; Unheated plate: colonies present

The table below shows the maximum number of spores from heated cultures plated which yielded zero colonies. In the case of B30, the germination assay was performed only once, and as visible in Fig. 2, contained viable, germinable spores. The numbers shown here are the highest number of spores plated, excluding B30. This means that we do not have data for higher concentrations of spores which would suggest any spore germinability. The mutant strain B42 contained germinable spores when 4x10⁷ were plated, but contrarily, zero germinable spores were found when 9.83x10⁷ spores were plated in another round of the germination assay.

Table 1: Germination rates of our mutant strains.