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ndoA (endoribonuclease toxin in Bacillus subtilis)

This coding region ndoA(also mazF, ydcE) encodes endoribonuclease EndoA, a toxic component of a type II toxin-antitoxin (TA) system. It encodes endoribonuclease toxin in Bacillus subtilis that is a UACAU‐specific mRNA interferase, cleavaging after the first U. Previous research has proved that its overexpression is toxic for cell growth and it functions as normal in Escherichia coli.

Usage and Biology

MazF is an mRNA interferase which cleaves mRNAs at a specific site, and ndoA, also called ydcE or mazF-bs shows a similar activity according to references given below. ndoA belongs to a type Ⅱ toxin-antitoxin system in Bacillus subtilis. There is great possibility that no antitoxin exists in C. glutamicum to inhibit EndoA encoded by ndoA. So it can be used to kill engineered C. glutamicum at a proper time.

Characterization

We aimed to test if the over-expression of the toxin gene ndoA from Bacillus subtilis can kill Corynebacterium glutamicum effectively as well.

Fig.1 Genetic circuit for ndoA verification

To characterize its killing effect, ndoA is inserted into the plasmid pXMJ19 to construct a circuit with tac promotor, lac operator, ndoA and terminator rrnB using the ClonExpress II one-step cloning kit (Vazyme Biotech, China). The expression vector is transformed into E. coli DH5α first and then into C. glutamicum by electroporation.

To design a quick quanlitative test, 'divided plate' assay was carried out at the beginning. We divided a plate with LB medium into four parts and use the spread plate method to see if ndoA can kill C. glutamicum and this killing effect is not caused by the toxicity of IPTG. Bacteria carrying empty vector was added into 1 Ep tube and bacteria carrying the gene circuit was added into 2 Ep tubes with 1.8 mL LB liquid containing 10 μg/mL chloramphenicol respectively. After incubating the culture in a shaker at 30 °C, 220 rpm until OD₆₀₀ reached 0.6, we prepared LB plates that were separated into four quarters marked A,B,C,D. We spread the diluted bacteria solution on the quarters respectively, and all plates were incubated at 30 °C for a certain time.

Figure 34 Divided Plate Assay
Quarter A: C. glutamicum carrying empty vector and 0.8mM IPTG was added; Quarter B: C. glutamicum carrying empty vector without adding 0.8mM IPTG; Quarter C: C. glutamicum carrying the vector pXMJ19-ndoA without adding 0.8mM IPTG; Quarter D: C. glutamicum carrying the vector pXMJ19-ndoA and 0.8mM IPTG was added.

Comparing Quarter A and Quarter B, we can see that the growth condition of the two is very similar, which means that the toxicity of 0.8mM IPTG is very low and it hardly kills C. glutamicum. Comparing Quarter C and Quarter D, we can see that there is apparently less bacteria survived in Quarter C, and the diameter of the colonies in Quarter C is rather small as well. As for the comparison between Quarter B and Quarter D, we assume that there is serious leaky expression of ndoA controlled by tac promoter in Quarter D, which deteriorates its growth condition. We repeated this experiments for 5 times and got nearly the same result. Hence, we can get the preliminary conclusion that ndoA does have a killing effect on C. glutamicum.

To give a further quantitative test, we carried out CFU assay to characterize the killing effect of ndoA instead of determining OD₆₀₀ in order to get rid of dead bacterial cells. CFU was quantified by counting the colonies on one plate and normalizing the number to volume of 1 mL culture. We added 0.8mM IPTG as OD₆₀₀ reached 0.6, and estimate CFU by spreading 25μL of the culture on 3 LB solid medium every hour after the induction with 0.8mM IPTG, and our results are as follows.

Figure 35 Results of the CFU Assay, plotted against induction

It is visually discovered that the number of colonies carrying pXMJ19-ndoA fell off in the presence of IPTG, while the same bacteria grew well without induction within the first four hours and then its CFU decrease probably due to the leaky expression. We can also see that the growth condition of the bacteria carrying the empty vector hasn’t been affected by 0.8mM IPTG, since its CFU curve increases as normal. We repeated this experiments for 3 times and all the curves show similar trends.

Therefore, we can finally come to the conclusion that the ndoA does have a killing effect on C. glutamicum, and can be used in our project as the toxin gene.

Sequence and Features