The deletion progress

Previous transcriptome data showed, that some genes are highly expressed in the MiniBacillus strain (Table 2). Part of these is the mhqNOP operon, which was over 4700-fold upregulated. This was caused by the deletion of the repressor MhqR, which normally regulates the resistance to quinones and diamide (Töwe et al., 2007; Reuß et al., 2017). However, the upregulated expression wastes a lot of energy and leads to an imbalance in the cell. Furthermore, the function of the mhqNOP genes is the protection against methyl-hydroquinone, which is unnecessary for the final MiniBacillus strain. Therefore, the plasmid pGP2093 was used to delete the mhqNOP operon in the strain PG18 with the marker-free deletion system as described in chapter 2.2.6., resulting in the strain PG29.

Table 2: Operons that are higher expressed in PG10, compared to Δ6

Operon Function Regulators Factor

mhqNOP Protection against hydroquinone MhqR (deleted) 4781 paiAB Control of intracellular polyamine

concentrations

420

The next step was to delete the paiAB operon with the plasmid pGP2094. This operon encodes for a spermine/ spermidine-N-acetyltransferase, which is also upregulated 420-fold. The resulting strain PG30 was further used to restore a point mutation in the pit gene, a low-affinity phosphate transporter. This point mutation was noticed in PG10 and leads probably to a reduction of phosphate uptake. However, this could be detrimental for the strain, since the reduced level of phosphate in the cell might activate a regulator system for phosphate metabolism, the PhoPR

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system. This leads to the activation of genes for the acquisition of phosphate and similar to the repression of the tagAB and tagDEFGH operons for the biosynthesis of teichoic acids (Prágai et al., 2004). Therefore, the pit point mutation was restored in strain PG31.

Since the essential or for the MiniBacillus necessary genes are scattered around the genome and the deletion regions become smaller, an approach to accelerate the deletion process might be the defragmentation. Functionally related genes, which should remain in the MiniBacillus, are clustered together in one locus. The native locus is then dispensable and can be deleted in one bigger deletion, instead of two smaller ones. This clustering can be done by introducing a second copy of the gene. In this case a glycolytic cassette should be introduced, containing the genes pgi, fbaA, ptsGHI. Pgi is the glucose-6-phosphate isomerase and FbaA the fructose-1,6-bisphophate aldolase and both enzymes are part of glycolysis, which plays a central role in the MiniBacillus blueprint. The operon ptsGHI encodes for the glucose phosphotransferase system which is responsible for the uptake of glucose in the cell. The introduction of this glycolytic cassette was shown to be functional in Zschiedrich (2014), however, the transfer of this construct would lead to the deletion of several genes including the gene nrnA. The deletion of nrnA, encoding for a nanoRNase, leads to the reduction of the competence in the MiniBacillus strain (Reuß, 2017).

Therefore, the glycolytic cassette was newly assembled and introduced next to nrnA, to sustain competence. This was done in two steps as shown in Figure 10. First the genes pgi and fbaA, together with a chloramphenicol resistance were introduced with a PCR product next to dnaE, leading to the deletion of the unknown gene ytrH (PG32). In the second step, the operon ptsGHI with a kanamycin resistance cassette was exchanged with the chloramphenicol resistance and the genes ytrI and ytzJ. The resulting strain PG33 was selected on plates with kanamycin and also tested for the loss of the chloramphenicol resistance. The genes pgi, fbaA and ptsGHI can now be deleted at their native locus to fasten the deletion process.

Figure 10: The two steps of the glycolytic cassette introduction into the MiniBacillus strain. First, the genes pgi and fbaA were integrated with a chloramphenicol cassette and in second step the genes ptsGHI were introduced with a kanamycin resistance.

fbaA

pgi fbaA cat^R ytrI ytzJ

dnaE

37 The following deletions from PG34 to 39 are deletions of genomic regions that are not necessary for the final MiniBacillus strain. The deletions are listed in Table 3, together with the plasmids that were used and the resulting genome size. The consequences of these deletions and the deletions that were already done before are discussed in chapter 3.1.3. The strains PG34, PG37 and PG39 were additionally analysed by WGS.

Table 3: The MiniBacillus deletion strains constructed in this work

strain Genome size

Deletion

plasmid Deletion

% reduction in comparison to wild type

WGS analysis

PG10 2759359 Reuß et al. (2017) 34.54 yes

PG18 2672270 Reuß (2017) 36.61 yes

PG29 2670199 pGP2093 ΔmhqNOP 36.66 no

PG30 2666896 pGP2094 yuzG-sufA 36.74 no

PG31 2666876 pJOE3256 pit point mutation 36.74 yes

PG32 2670874 PCR product insertion pgi, fbaA, cat; Deletion ytrH

36.64 no

PG33 2674507 PCR product insertion ptsGHI, kan; Deletion ytrI, ytzJ, cat

36.56 no

PG34 2652827 pGP2098 ycgQ-yckE 37.07 yes

PG35 2635284 pGP2088 yvaM-yvbK 37.49 no

PG36 2622356 pGP2073 nhaX-yhaX 37.79 no

PG37 2587747 pGP2270 glpQ-ycbK 38.62 yes

PG38 2554562 pGP2282 yqjF-yqjG 39.40 no

PG39 2507732 pGP2283 yddN-ydfM 40.51 yes

The final MiniBacillus strain of this work PG39 has a genome size of about 2.5 Mbp, which corresponds to a genome reduction of 40.51% in comparison to the wild type strain 168. This is the greatest reduction of the B. subtilis genome published so far.

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