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Review
. 2016 Sep 28;80(4):955-987.
doi: 10.1128/MMBR.00029-16. Print 2016 Dec.

The Blueprint of a Minimal Cell: MiniBacillus

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Free PMC article
Review

The Blueprint of a Minimal Cell: MiniBacillus

Daniel R Reuß et al. Microbiol Mol Biol Rev. .
Free PMC article

Abstract

Bacillus subtilis is one of the best-studied organisms. Due to the broad knowledge and annotation and the well-developed genetic system, this bacterium is an excellent starting point for genome minimization with the aim of constructing a minimal cell. We have analyzed the genome of B. subtilis and selected all genes that are required to allow life in complex medium at 37°C. This selection is based on the known information on essential genes and functions as well as on gene and protein expression data and gene conservation. The list presented here includes 523 and 119 genes coding for proteins and RNAs, respectively. These proteins and RNAs are required for the basic functions of life in information processing (replication and chromosome maintenance, transcription, translation, protein folding, and secretion), metabolism, cell division, and the integrity of the minimal cell. The completeness of the selected metabolic pathways, reactions, and enzymes was verified by the development of a model of metabolism of the minimal cell. A comparison of the MiniBacillus genome to the recently reported designed minimal genome of Mycoplasma mycoides JCVI-syn3.0 indicates excellent agreement in the information-processing pathways, whereas each species has a metabolism that reflects specific evolution and adaptation. The blueprint of MiniBacillus presented here serves as the starting point for a successive reduction of the B. subtilis genome.

Figures

FIG 1
FIG 1
Outline of the metabolic model of the minimal cell. The model gives an overview of the metabolic pathways of the intended minimal organism. Functionally related pathways are grouped in boxes. Details on all reactions and enzymes are provided in Fig. 2 to 11. DHAP, dihydroxyacetone phosphate; G3P, glycerol-3-phosphate; AA, amino acid; THF, tetrahydrofolate; 2-OG, 2-oxoglutarate; GAP, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; PLP, pyridoxal phosphate; DHF, 7,8-dihydrofolate; pABA, 4-aminobenzoate; FMN, flavin mononucleotide; PRPP, phosphoribosyl pyrophosphate; E4P, erythrose-4-phosphate; 3PG, 3-phosphoglycerate; PP, pyrophosphate; F6P, fructose-6-phosphate.
FIG 2
FIG 2
Miscellaneous pathways. The model shows the uptake of metal ions and inorganic phosphate (Pi) and reactions for protective functions, for the generation of phosphatidic acid, and for the synthesis and degradation of c-di-AMP. Finally, protein secretion is included. The metabolic intermediates diacylglycerol (Fig. 6) and phosphatidic acid (Fig. 6) that occur in other pathways are labeled in blue. P, protein.
FIG 3
FIG 3
Central carbon metabolism and energy conservation. (A) Glycolytic and pentose phosphate pathways. (B) The respiratory chain and ATPase. (C) The transhydrogenase cycle for balancing NADPH2. (D) Recycling of acetate derived from cell wall metabolism (Fig. 10). The following metabolic intermediates that occur in other pathways are labeled in blue: phosphoenolpyruvate (PEP) (Fig. 4 and 10), glucose-6-phosphate (Glucose-6-P) (Fig. 6), fructose-6-phosphate (Fig. 10), glyceraldehyde-3-phosphate (GAP) (Fig. 7 and 10), dihydroxyacetone phosphate (DHAP) (Fig. 6), 3-P-Glycerate (Fig. 4), pyruvate (Fig. 4 and 8 to 10), acetyl-CoA (Fig. 6 and 10), ribose-5-phosphate (Fig. 5 and 7), erythrose-4-phosphate (Fig. 4), menaquinone/menaquinol (MQ) (Fig. 9), heme (Fig. 8), acetate (Fig. 10), and coenzyme A (Fig. 6 and 10). FBP, fructose 1,6-bisphosphate.
FIG 4
FIG 4
Acquisition of amino acids and charging of tRNAs. The following metabolic intermediates that occur in other pathways are labeled in blue: 3-P-Glycerate (Fig. 3), tetrahydrofolate (THF) (Fig. 7 and 8), methyltetrahydrofolate (Methyl-THF) (Fig. 5 and 7), pyruvate (Fig. 3 and 8 to 10), 2-oxoglutarate (2-OG) (Fig. 9), erythrose-4-phosphate (Fig. 3), phosphoenolpyruvate (PEP) (Fig. 3 and 10), and chorismate (Fig. 8 and 9). DAHP, 3-deoxy-d-arabino-hept-2-ulosonate 7-phosphate; EPSP, 5-O-(1-carboxyvinyl)-3-phosphoshikimate; AA, amino acid.
FIG 5
FIG 5
Acquisition of nucleotides. The following metabolic intermediates that occur in other pathways are labeled in blue: methyltetrahydrofolate (Methyl-THF) (Fig. 4 and 7), ribose-5-phosphate (Fig. 3 and 7), phosphoribosyl pyrophosphate (PRPP) (Fig. 7), and formyltetrahydrofolate (Formyl-THF) (Fig. 8). AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide.
FIG 6
FIG 6
(A) Biosynthesis of lipids. The enzyme required for the conversion of phosphatidylglycerol phosphate to phosphatidylglycerol is unknown. (B) Biosynthesis of lipoteichoic acids. The enzyme required for the export of diacyl-3-(diglucopyranosyl)-glycerol is unknown. The following metabolic intermediates that occur in other pathways are labeled in blue: acetyl-CoA (Fig. 3 and 10), CoA (Fig. 3, 7, and 9), glycerol-3-phosphate (G3P) (Fig. 11), dihydroxyacetone phosphate (DHAP) (Fig. 3), phosphatidylglycerol (this figure), glucose-6-phosphate (Fig. 3), diacylglycerol (Fig. 2), and phosphatidic acid (Fig. 2).
FIG 7
FIG 7
Acquisition of cofactors and biosynthesis of iron-sulfur clusters. The following metabolic intermediates that occur in other pathways are labeled in blue: glyceraldehyde-3-phosphate (GAP) (Fig. 3 and 10), ribose-5-phosphate (Fig. 3 and 5), S-adenosylmethionine (SAM) (this figure), phosphoribosyl pyrophosphate (PRPP) (Fig. 5), methyltetrahydrofolate (Methyl-THF) (Fig. 4 and 5), tetrahydrofolate (THF) (Fig. 4 and 8), and coenzyme A (CoA) (Fig. 3, 6, and 9). PLP, pyridoxal phosphate; FMN, flavin mononucleotide; FeS, iron-sulfur cluster; 3-Methyl-2-OB, 3-methyl-2-oxobutanoate.
FIG 8
FIG 8
Acquisition of cofactors. The following met1abolic intermediates that occur in other pathways are labeled in blue: chorismate (Fig. 4 and 9), 4-aminobenzoate (pABA) (this figure), pyruvate (Fig. 3, 4, 9, and 10), tetrahydrofolate (THF) (Fig. 4 and 7), formyltetrahydrofolate (Formyl-THF) (Fig. 5), farnesyl pyrophosphate (farnesyl-PP) (Fig. 10), and heme O (Fig. 3). DHF, 7,8-dihydrofolate.
FIG 9
FIG 9
Acquisition of cofactors. The following metabolic intermediates that occur in other pathways are labeled in blue: chorismate (Fig. 4 and 8), 2-oxoglutarate (2-OG) (Fig. 4), pyruvate (Fig. 3, 4, 8, and 10), coenzyme A (CoA) (Fig. 3, 6, and 7), and menaquinone (Fig. 3).
FIG 10
FIG 10
Biosynthesis of the cell wall. The following metabolic intermediates that occur in other pathways are labeled in blue: pyruvate (Fig. 3, 4, 8, and 9), glyceraldehyde-3-phosphate (GAP) (Fig. 3 and 7), isopentenyl pyrophosphate (isopentenyl-PP) (this figure), farnesyl pyrophosphate (farnesyl-PP) (Fig. 8), undecaprenyl phosphate (Fig. 11), fructose-6-phosphate (Fig. 3), acetyl-CoA (Fig. 3 and 6), UDP-N-acetylglucosamine (UDP-GlcNAc) (Fig. 11), phosphoenol pyrophosphate (PEP) (Fig. 3 and 4), and acetate (Fig. 3). UDP-MurNAc, UDP-N-acetylmuramic acid; DAP, diaminopimelate.
FIG 11
FIG 11
Biosynthesis of wall teichoic acids. The following metabolic intermediates that occur in other pathways are labeled in blue: undecaprenyl phosphate (Fig. 10), UDP N-acetylglucosamine (UDP-GlcNAc) (Fig. 10), CDP-glycerol (this figure), and glycerol-3-phosphate (G3P) (Fig. 6). UDP-ManNAc, UDP-N-acetylmannosamine.
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