The Blueprint of a Minimal Cell: MiniBacillus

Daniel R Reuß; Fabian M Commichau; Jan Gundlach; Bingyao Zhu; Jörg Stülke

doi:10.1128/MMBR.00029-16

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

The Blueprint of a Minimal Cell: MiniBacillus

Daniel R Reuß ¹, Fabian M Commichau ¹, Jan Gundlach ¹, Bingyao Zhu ¹, Jörg Stülke ^1,^✉

PMCID: PMC5116877 PMID: 27681641

SUMMARY

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.

INTRODUCTION

Three technological revolutions dramatically changed our view of biology. The genomic revolution gives us access to genome sequences of any organism of interest at low cost. The analytical revolution, especially with respect to mass spectrometry, allows us not only to detect the presence and the fluxes of any molecule in the cell but also to study its precise concentration under any desired condition. Last but not least, the informatics revolution paves the way for the evaluation of the tremendous data sets and for the generation of meaningful models and predictions of cellular behavior. However, even knowledge of all components of a cell and of their precise concentrations does not give us a complete understanding of a living cell. For this, we have to consider all the functional interactions between different biological molecules and the dynamics of both the molecules and their interactions.

The complexity of all naturally existing organisms still precludes a deep understanding of the functions of all components of a cell and their interactions. Even small organisms such as bacteria are too complex to understand all processes in their cells. This is even the case for bacteria with naturally minimal genomes, as found in the genus Mycoplasma. These bacteria may have as few as 482 protein-coding genes and are still capable of independent life in the absence of any host cells. However, the functions of many Mycoplasma genes are so far unknown, and not the full gene set is essential, indicating that we are far from a full picture of these bacteria despite tremendous efforts in their analysis (1 –8).

These limitations in our understanding of natural organisms call for a reduction of complexity: the creation of cells with a defined set of genes. Such cells can be obtained by applying either bottom-up or top-down approaches. The former approach has so far been pursued with the chemical synthesis of a bacterial genome and its application to create a semiartificial cell (9, 10). In this case, a known genome was reduced and transplanted into a closely related host cell. With only 473 protein-coding genes, the recently achieved Mycoplasma mycoides JCVI-syn3.0 minigenome is so far the smallest semiartificially designed organism. Importantly, about one-third of the proteins in this minimal cell are of unknown function (10). Moreover, there have been attempts to create so-called protocells, which are lipid vesicles that contain genetic material and/or enzymes (11 –14). Even though protocells do not allow recapitulation of the evolutionary emergence of life, they are well-suited systems to study the physical and biochemical properties of basic cellular processes such as self-reproduction, permeability, enzymatic replication, and Darwinian evolution (15 –17). Reduction of complexity can also be achieved by a top-down approach that starts with existing bacteria and aims at consecutively reducing their complexity. This approach is, of course, very time-consuming; on the other hand, it allows advancing from step to step. Moreover, this iterative process of genome reduction allows the immediate discovery of possible problems and, thus, finding appropriate solutions. Genome reduction is a common theme in synthetic biology, not only for pure scientific curiosity but also from an industrial point of view to create workhorses for biotechnology. Ongoing projects of genome reduction have been reported for several intensively studied bacteria such as Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Pseudomonas putida, and Streptomyces avermitilis (18 –27; for a review, see reference 28) as well as for yeast (29). All these projects are still far from the final goal, the minimal cell.

With the progress of genome reduction, it is necessary to define the set of genes that should be part of the final minimal genome. It is obvious that such a set of genes is determined by several factors, including the intended lifestyle of the final minimal cell, but also by the general biology of the organism that is to be reduced. Conceivably, a eukaryotic yeast cell will still contain a nucleus even at a late genome reduction stage. Similarly, the bacteria mentioned above differ strongly in their cellular organizations. For example, M. mycoides does not possess a cell wall, while the cell wall is differently structured yet essential in B. subtilis, C. glutamicum, and E. coli. In this work, we aim at defining the set of genes that is required for the life of a minimal cell based on B. subtilis. For several reasons, this bacterium is particularly well suited for genome minimization approaches. First, B. subtilis is one of the most intensively studied organisms, with extensive genome annotation and excellent knowledge of the major cellular processes. Second, the elaborated genetic system for B. subtilis makes all kinds of genetic manipulations very easy (see below). Finally, B. subtilis is one of the major organisms in biotechnology, suggesting that genome-reduced strains may also serve as a chassis for novel applications.

CONSIDERATIONS FOR THE DEFINITION OF THE GENE SET FOR A MINIMAL CELL

Several independent sets of information serve as the basis to define which genes are required for a viable minimal cell. First of all, this is the set of essential genes. These genes were identified for B. subtilis in 2003 (30). In addition, large dispensable regions of the chromosome have been studied, resulting in the identification of novel essential and coessential genes (31). A recent reevaluation of the essential genes of B. subtilis revealed that several metabolic genes involved in glycolysis and the tricarboxylic acid cycle originally listed as essential could be removed from the list. With the exception of the ylaN gene, all other genes of unknown function could also be removed from the list (32, 33). Moreover, recent studies indicated that the ycgG and yfkN genes as well as the rny gene, encoding RNase Y, are also dispensable (28, 34; our unpublished data). The current list of 251 essential protein-coding genes is available in the SubtiWiki database (http://subtiwiki.uni-goettingen.de/wiki/index.php/Essential_genes) (33).

The essential genes are by definition only those genes that cannot be deleted as single genes under defined optimal growth conditions (for B. subtilis, lysogeny broth [LB] with glucose at 37°C). Moreover, a recent knockdown study of essential B. subtilis genes showed that the encoded proteins are also very important for outgrowth from stationary phase, adding another level of relevance (35). However, many genes are redundant, and cellular functions can be achieved in completely different ways. The former is the case for DNA polymerase I (PolA) and its paralog YpcP or the diadenylate cyclase CdaA and one of the paralogs DisA and CdaS (36, 37). Moreover, the same function may even be fulfilled by unrelated proteins, as observed for the membrane anchors for the Z-ring protein for cell division, FtsZ. In E. coli, the essential FtsA protein serves as a membrane anchor for FtsZ. Why FtsA is nonessential in B. subtilis has been enigmatic for a long time. Only the discovery of the unrelated alternative membrane anchor SepF provided the answer (38). Finally, alternative pathways may lead to the same results. This is obvious in the acquisition of cellular building blocks such as amino acids and nucleotides. These metabolites can be either synthesized in the cell or taken up from the medium. In any case, none of the involved genes would be classified as being essential. In all these cases, a decision has to be made regarding which of the possible alternatives will be included in the minimal gene set. Accordingly, the gene complement of a minimal organism has to be designed according to essential functions.

If B. subtilis possesses multiple genes for the same function, one of them has to be selected. The criteria for the selection applied in this study are as follows. (i) The final number of genes should be as small as possible. Therefore, it seems reasonable to include transporters rather than biosynthetic genes for the acquisition of building blocks whenever possible. (ii) In some functional categories, such as cell division, the deletion of a gene may have only a mild effect; however, combination with the deletion of a second, sometimes functionally unrelated gene may be lethal (see “Cell Division,” below, for details). Therefore, such synthetic lethalities have to be considered. (iii) Prior decisions will have an impact on later selections. This is the case, for example, for cell wall-biosynthetic proteins (see below). (iv) Both gene expression and protein levels have been extensively studied in B. subtilis, and all these data are accessible in the SubtiWiki database (33, 39 –41). In case of doubt, the most strongly expressed protein has been chosen. (v) Finally, conservation of genes served as a criterion. More strongly conserved genes were preferred over less conserved genes. In this respect, gene conservation and essentiality in genome-reduced Mycoplasma and other mollicutes species and the inclusion of genes in the genome of M. mycoides JCVI-syn3.0 had a high priority (8, 10, 42).

In many cases, it is not known whether a gene is truly required in the context of a minimal cell. In particular, this is the case for functions involved in RNA modification. In these cases, expression levels and gene conservation were valuable clues for deciding whether a gene should be included in the minimal gene list or not. Based on the list of the most abundant proteins (see http://subtiwiki.uni-goettingen.de/wiki/index.php/Most_abundant_proteins) (33, 43), we have decided whether there is a good reason to keep the corresponding genes or not. As an example, highly abundant enzymes required for amino acid biosynthesis were selected for deletion, whereas RNA chaperones were added. Similarly, genes conserved both in all mollicutes and in B. subtilis were regarded as being highly relevant for a minimal organism based on B. subtilis.

THE GENETIC COMPLEMENT OF A MINIMAL CELL

Based on the considerations explained above, we have selected 523 and 119 protein- and RNA-coding genes, respectively, as being important for a minimal organism that is capable of growing in LB medium supplemented with glucose at 37°C. Moreover, the growth and physiology of the minimal cell should be comparable to those of B. subtilis wild-type cells. B. subtilis has a generation time of about 20 min, whereas natural minimal organisms like M. mycoides and Mycoplasma pneumoniae divide in about 1 and 30 h, respectively. The minimal organism M. mycoides JCVI-syn3.0 has a generation time of 180 min (10, 44). This slow growth of Mycoplasma cells is another reason to rely on B. subtilis as the basis for a minimal cell. Most likely, a reduction of the growth rate has to be expected; however, the set of genes suggested in this study should allow generation times of <1 h. As a consequence, the number of genes included in the list is much larger than that of essential genes in B. subtilis 168. Moreover, not all essential genes are required for a minimal cell since several essential genes fulfill protective functions that are dispensable if, e.g., prophages have been deleted (32).

The genes of this minimal set satisfy all essential functions of the cell, such as information processing (DNA replication, transcription, and translation), metabolic pathways (metabolism of building blocks and cofactors and acquisition of ions, etc.), as well as cell division and integrity. Interestingly, there is a very good match between the genes in the MiniBacillus minimal gene set and those of M. mycoides JCVI-syn3.0 as far as information processing is concerned. In contrast, the two lists show only little overlap of genes required for metabolism, cell division, and protective functions. An overview of the set of genes required for MiniBacillus is provided in Table 1. A model of the metabolism of the minimal cell is outlined in Fig. 1, and details that include all metabolic pathways, reactions, and enzymes are provided in Fig. 2 to 11. Detailed information on each individual gene can be found in Tables 2 and 3 and Table S1 in the supplemental material. Table 2 also shows whether the components proposed to be important in the frame of a minimal B. subtilis genome are also present in the recently published minimal strain M. mycoides JCVI-syn3.0.

TABLE 1.

Overview of the genetic complement of a minimal B. subtilis cell

Function	No. of proteins (no. of essential proteins)^a	No. of RNA genes (no. of essential genes)^a	Figure(s)
Information processing	197 (125)	119 (2)
DNA replication	18 (15)
Chromosome maintenance	13 (9)
Transcription	8 (5)
RNA folding and degradation	6 (1)
Aminoacyl-tRNA synthetases	24 (23)		4
Ribosomal proteins	53 (35)
rRNA and tRNA		116 (0)
rRNA/tRNA maturation and modification	31 (13)	1 (1)
Ribosome maturation and assembly	9 (6)
Translation factors	11 (9)
Translation/other	5 (2)	1 (0)
Protein secretion	12 (5)	1 (1)	2
Proteolysis, protein quality control, chaperones	7 (2)
Metabolism	218 (59)
Central carbon metabolism	26 (4)		3
Respiration/energy	16 (2)		3
Amino acids	30 (1)^b		3
Nucleotides/phosphate	36 (11)		2, 5
Lipids	19 (17)		6
Cofactors	62 (14)^b
General components of ECF transporters	3 (0)^b		4, 7
NAD	5 (4)		7
FAD	2 (1)^b		7
Pyridoxal phosphate	2 (0)		7
Biotin	1 (0)^b		7
Thiamine	2 (0)^b		7
Lipoate	4 (1)		7
Coenzyme A	9 (1)		7
S-Adenosylmethionine	1 (1)		7
Folate	13 (1)		8
Heme	12 (0)		8
Menaquinone	8 (5)		9
Metals/iron-sulfur clusters	29 (10)		2, 7
Cell division	81 (52)
Cell wall synthesis	55 (41)
Amino acid precursor	11 (10)		10
Undecaprenyl phosphate	13 (10)		10
Lipid II biosynthesis	12 (11)		10
Peptidoglycan polymerization	5 (1)		10
Wall teichoic acid	9 (8)		11
Lipoteichoic acid	5 (1)		6
Coordination	22 (9)
Signaling	4 (2)		2
Integrity of the cell	16 (5)
Protection	8 (4)		2
Repair/genome integrity	8 (1)
Other/unknown	11 (2)
Total minimal genome	523 (243)	119 (2)

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The numbers of proteins and RNAs required for each function are listed. Numbers in parentheses indicate the numbers of proteins and RNAs that are essential in the context of B. subtilis 168.

Tryptophan, riboflavin, biotin, and thiamine are transported by transporters of the ECF (energy-coupling factor) family. The three general components are shared among all these transporters. They are listed separately with the cofactors.

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 — Miscellaneous pathways. The model shows the uptake of metal ions and inorganic phosphate (P_i) 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 — 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 — 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 — 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 — (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 — 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 — 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 — 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 — 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 — 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.

TABLE 2.

The complete gene set of MiniBacillus^e

Gene	BSU no.^a	Essential^b	Syn3.0^c	EC no.	PDB accession no.	Organism^d	Function(s)
Information
DNA replication
dnaA	BSU00010	Yes	Yes		2Z4R	Thermotoga maritima	Replication initiation protein
dnaB	BSU28990	Yes					Initiation of chromosome replication
dnaC	BSU40440	Yes	Yes	3.6.4.12	2VYE	Geobacillus kaustophilus	Replicative DNA helicase
dnaD	BSU22350	Yes			2V79	B. subtilis	Initiation of chromosome replication
dnaE	BSU29230	Yes	Yes	2.7.7.7	3E0D	Thermus aquaticus	DNA polymerase III (alpha subunit)
dnaG	BSU25210	Yes	Yes	2.7.7.-	4E2K	S. aureus	DNA primase
dnaI	BSU28980	Yes	Yes		4M4W	B. subtilis	Primosome component (helicase loader)
dnaN	BSU00020	Yes	Yes	2.7.7.7	4TR6	B. subtilis	DNA polymerase III (beta subunit), beta clamp
dnaX	BSU00190	Yes	Yes	2.7.7.7	1JR3	E. coli	DNA polymerase III (gamma and tau subunits)
holA	BSU25560	Yes	Yes	2.7.7.7	3ZH9	B. subtilis	DNA polymerase III, delta subunit
holB	BSU00310	Yes		2.7.7.7	1NJG	E. coli	DNA polymerase III (delta subunit)
ligA	BSU06620	Yes	Yes	6.5.1.2	2OWO	E. coli	DNA ligase (NAD dependent)
priA	BSU15710	Yes		3.6.4.-	4NL4	Klebsiella pneumoniae	Primosomal replication factor Y
polC	BSU16580	Yes	Yes	2.7.7.7	3F2B	G. kaustophilus	DNA polymerase III (alpha subunit)
rtp	BSU18490	No			1BM9	B. subtilis	Replication terminator protein
ssbA	BSU40900	Yes	Yes		3VDY	B. subtilis	Single-strand DNA-binding protein
yabA	BSU00330	No			5DOL	B. subtilis	Inhibitor of DnaA oligomerization
polA	BSU29090	No	Yes	2.7.7.7	1BGX	T. aquaticus	DNA polymerase I
Chromosome maintenance
scpA	BSU23220	Yes			3ZGX	B. subtilis	DNA segregation/condensation protein
scpB	BSU23210	Yes	Yes		3W6J	Geobacillus stearothermophilus	DNA segregation/condensation protein
smc	BSU15940	Yes	Yes		3ZGX	B. subtilis	SMC protein
parE	BSU18090	Yes	Yes	5.99.1.-	4I3H	Streptococcus pneumoniae	Subunit of DNA topoisomerase IV
parC	BSU18100	Yes	Yes	5.99.1.-	2INR	S. aureus	Subunit of DNA topoisomerase IV
spoIIIE	BSU16800	No			2IUT	Pseudomonas aeruginosa	ATP-dependent DNA translocase
sftA	BSU29805	No			2IUT	P. aeruginosa	DNA translocase
codV	BSU16140	No			1A0P	E. coli	Site-specific integrase/recombinase
ripX	BSU23510	No			1A0P	E. coli	Site-specific integrase/recombinase
gyrB	BSU00060	Yes	Yes	5.99.1.3	4I3H	S. pneumoniae	DNA gyrase (subunit B)
gyrA	BSU00070	Yes	Yes	5.99.1.3	4DDQ	B. subtilis	DNA gyrase (subunit A)
topA	BSU16120	Yes	Yes	5.99.1.2	4RUL	E. coli	DNA topoisomerase I
hbs	BSU22790	Yes			1HUE	G. stearothermophilus	Nonspecific DNA-binding protein HBsu
Transcription
rpoA	BSU01430	Yes	Yes	2.7.7.6	3IYD	E. coli	RNA polymerase alpha subunit
rpoB	BSU01070	Yes	Yes	2.7.7.6	3IYD	E. coli	RNA polymerase beta subunit
rpoC	BSU01080	Yes	Yes	2.7.7.6	3IYD	E. coli	RNA polymerase beta′ subunit
sigA	BSU25200	Yes	Yes		3IYD	E. coli	RNA polymerase sigma factor SigA
rpoE	BSU37160	No		2.7.7.6	2KRC	B. subtilis	RNA polymerase delta subunit
helD	BSU33450	No		3.6.4.12			DNA 3′–5′ helicase IV
greA	BSU27320	No	Yes		1GRJ	E. coli	Transcription elongation factor
nusA	BSU16600	Yes	Yes		1HH2	T. maritima	Transcription termination factor
RNA folding and degradation
cspD	BSU21930	No			1C9O	Bacillus caldolyticus	Cold shock protein
cspB	BSU09100	No			2ES2	B. subtilis	Major cold shock protein
rny	BSU16960	No	Yes	3.1.4.16			RNase Y
rnjA	BSU14530	Yes	Yes		3ZQ4	B. subtilis	RNase J1
pnpA	BSU16690	No		2.7.7.8	3CDI	E. coli	Polynucleotide phosphorylase
nrnA	BSU29250	No	Yes	3.1.3.7	3DEV	Staphylococcus haemolyticus	Oligoribonuclease (nano-RNase)
Aminoacyl tRNA synthetases
alaS	BSU27410	Yes	Yes	6.1.1.7	3HTZ	E. coli	Alanine-tRNA synthetase
argS	BSU37330	Yes	Yes	6.1.1.19	3FNR	Campylobacter jejuni	Arginyl-tRNA synthetase, universally conserved protein
asnS	BSU22360	Yes	Yes	6.1.1.22	1X54	Pyrococcus horikoshii	Asparagyl-tRNA synthetase
aspS	BSU27550	Yes	Yes	6.1.1.12	1EQR	E. coli	Aspartyl-tRNA synthetase
cysS	BSU00940	Yes	Yes	6.1.1.16	3TQO	Coxiella burnetii	Cysteine-tRNA synthetase
gatC	BSU06670	Yes		6.3.5.7	2DF4	S. aureus	Production of glutamyl-tRNA^Gln
gatA	BSU06680	Yes	Yes	6.3.5.7	2DF4	S. aureus	Production of glutamyl-tRNA^Gln
gatB	BSU06690	Yes	Yes	6.3.5.7	2DF4	S. aureus	Production of glutamyl-tRNA^Gln
gltX	BSU00920	Yes	Yes	6.1.1.17	2O5R	T. maritima	Glutamyl-tRNA synthetase, universally conserved protein
glyS	BSU25260	Yes		6.1.1.14			Glycyl-tRNA synthetase (beta subunit)
glyQ	BSU25270	Yes		6.1.1.14	1J5W	T. maritima	Glycyl-tRNA synthetase (alpha subunit)
hisS	BSU27560	Yes	Yes	6.1.1.21	1QE0	S. aureus	Histidyl-tRNA synthetase
ileS	BSU15430	Yes	Yes	6.1.1.5	1QU2	S. aureus	Isoleucyl-tRNA synthetase
leuS	BSU30320	Yes	Yes	6.1.1.4	1OBH	Thermus thermophilus	Leucyl-tRNA synthetase
lysS	BSU00820	Yes	Yes	6.1.1.6	3E9H	G. stearothermophilus	Lysyl-tRNA synthetase
metS	BSU00380	Yes	Yes	6.1.1.10	4QRD	S. aureus	Methionyl-tRNA synthetase
pheT	BSU28630	Yes	Yes	6.1.1.20	2RHS	S. haemolyticus	Phenylalanyl-tRNA synthetase (beta subunit)
pheS	BSU28640	Yes	Yes	6.1.1.20	2RHQ	S. haemolyticus	Phenylalanyl-tRNA synthetase (alpha subunit), universally conserved protein
proS	BSU16570	Yes		6.1.1.15	2J3L	Enterococcus faecalis	Prolyl-tRNA synthetase
serS	BSU00130	Yes	Yes	6.1.1.11	2DQ3	Aquifex aeolicus	Seryl-tRNA synthetase
thrS	BSU28950	No	Yes	6.1.1.3	1NYQ	S. aureus	Threonyl-tRNA synthetase (major)
trpS	BSU11420	Yes	Yes	6.1.1.2	3PRH	B. subtilis	Tryptophanyl-tRNA synthetase
tyrS	BSU29670	Yes	Yes	6.1.1.1	2TS1	G. stearothermophilus	Tyrosyl-tRNA synthetase (major)
valS	BSU28090	Yes	Yes	6.1.1.9	1GAX	T. thermophilus	Valyl-tRNA synthetase
Ribosomal proteins
rplA	BSU01030	No	Yes		3J9W	B. subtilis	Ribosomal protein L1
rplB	BSU01190	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L2
rplC	BSU01160	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L3
rplD	BSU01170	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L4
rplE	BSU01280	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L5
rplF	BSU01310	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L6
rplI	BSU40500	No	Yes		3J9W	B. subtilis	Ribosomal protein L9
rplJ	BSU01040	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L10
rplK	BSU01020	No	Yes		3J9W	B. subtilis	Ribosomal protein L11
rplL	BSU01050	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L12
rplM	BSU01490	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L13
rplN	BSU01260	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L14
rplO	BSU01350	No	Yes		3J9W	B. subtilis	Ribosomal protein L15
rplP	BSU01230	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L16
rplQ	BSU01440	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L17
rplR	BSU01320	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L18
rplS	BSU16040	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L19
rplT	BSU28850	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L20
rplU	BSU27960	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L21
rplV	BSU01210	No	Yes		3J9W	B. subtilis	Ribosomal protein L22
rplW	BSU01180	No	Yes		3J9W	B. subtilis	Ribosomal protein L23
rplX	BSU01270	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L24
rpmA	BSU27940	Yes	Yes		3J9W	B. subtilis	Ribosomal protein L27
rpmB	BSU15820	No			3J9W	B. subtilis	Ribosomal protein L28
rpmC	BSU01240	No			3J9W	B. subtilis	Ribosomal protein L29
rpmD	BSU01340	Yes			3J9W	B. subtilis	Ribosomal protein L30
rpmE	BSU37070	No			3J9W	B. subtilis	Ribosomal protein L31
rpmF	BSU15080	No			3J9W	B. subtilis	Ribosomal protein L32
rpmGA	BSU24900	No			3J9W	B. subtilis	Ribosomal protein L33a
rpmGB	BSU00990	No			3J9W	B. subtilis	Ribosomal protein L33b
rpmH	BSU41060	No			3J9W	B. subtilis	Ribosomal protein L34
rpmI	BSU28860	No			3J9W	B. subtilis	Ribosomal protein L35
rpmJ	BSU01400	No	Yes		3J9W	B. subtilis	Ribosomal protein L36
rpsB	BSU16490	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S2
rpsC	BSU01220	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S3
rpsD	BSU29660	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S4
rpsE	BSU01330	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S5
rpsF	BSU40910	No	Yes		3J9W	B. subtilis	Ribosomal protein S6
rpsG	BSU01110	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S7
rpsH	BSU01300	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S8
rpsI	BSU01500	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S9
rpsJ	BSU01150	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S10
rpsK	BSU01420	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S11
rpsL	BSU01100	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S12
rpsM	BSU01410	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S13
rpsN	BSU01290	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S14
rpsO	BSU16680	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S15
rpsP	BSU15990	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S16
rpsQ	BSU01250	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S17
rpsR	BSU40890	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S18
rpsS	BSU01200	Yes	Yes		3J9W	B. subtilis	Ribosomal protein S19
rpsT	BSU25550	No			3J9W	B. subtilis	Ribosomal protein S20
rpsU	BSU25410	No			3J9W	B. subtilis	Ribosomal protein S21
rRNA/tRNA maturation and modification
rnpA	BSU41050	Yes	Yes	3.1.26.5	3Q1R	T. maritima	Protein component of RNase P
rnpB	BSU_misc_RNA_35	Yes			3Q1R	T. maritima	RNA component of RNase P
rnz	BSU23840	Yes		3.1.26.11	4GCW	B. subtilis	RNase Z
rph	BSU28370	No		2.7.7.56	1OYP	B. subtilis	RNase PH
rbfA	BSU16650	No			1JOS	Haemophilus influenzae	Ribosome-binding factor A
rimM	BSU16020	No			3H9N	H. influenzae	16S rRNA-processing protein, RNase
cca	BSU22450	Yes		2.7.7.25	1MIY	G. stearothermophilus	tRNA nucleotidyltransferase
fmt	BSU15730	Yes	Yes	2.1.2.9	4IQF	Bacillus anthracis	Methionyl-tRNA formyltransferase
folD	BSU24310	Yes	Yes	1.5.1.5	1B0A	E. coli	Methylenetetrahydrofolate dehydrogenase
rlmCD	BSU06730	No		2.1.1.190	2BH2	E. coli	rRNA methyltransferase
ysgA	BSU28650	Yes	Yes	2.1.1.-	4X3M	T. thermophilus	Similar to rRNA methylase
mraW	BSU15140	No	Yes	2.1.1.199	1WG8	T. thermophilus	SAM-dependent methyltransferase
cspR	BSU08930	Yes		2.1.1.-	4PZK	B. anthracis	Similar to tRNA(Um34/Cm34) methyltransferase
trmD	BSU16030	Yes	Yes	2.1.1.31	3KY7	S. aureus	tRNA methyltransferase
trmU	BSU27500	Yes	Yes	2.8.1.-	2HMA	S. pneumoniae	tRNA(5-methylaminomethyl-2-thiouridylate) methyltransferase
yrvO	BSU27510	Yes		2.8.1.7	1P3W	E. coli	Cysteine desulfurase
yacO	BSU00960	No	Yes	2.1.1.-	1GZ0	E. coli	Putative 23S rRNA methyltransferase
ksgA	BSU00420	No	Yes	2.1.1.-	3FUU	T. thermophilus	rRNA adenine dimethyltransferase
rluB	BSU23160	No	Yes	5.4.99.22	4LAB	E. coli	Pseudouridine synthase
ypuI	BSU23200	No					rRNA pseudouridine 2633 synthase
tilS	BSU00670	Yes	Yes	6.3.4.19	3A2K	G. kaustophilus	tRNA^Ile lysidine synthetase
tsaB	BSU05920	Yes			2A6A	T. maritima	Threonyl carbamoyl adenosine (t6A) modification of tRNAs that pair with ANN codons in mRNA
tsaD	BSU05940	Yes	Yes	2.3.1.234	3ZET	Salmonella enterica serovar Typhimurium	Threonyl carbamoyl adenosine (t6A) modification of tRNAs that pair with ANN codons in mRNA, universally conserved protein
tsaC	BSU36950	No		2.7.7.87	3AJE	Sulfolobus tokodaii	l-Threonyl carbamoyl AMP synthase, biosynthesis of the hypermodified base threonyl carbamoyl adenosine [t(6)A]
gidA	BSU41010	No	Yes		3CP2	E. coli	tRNA uridine 5-carboxymethyl-aminomethyl modification enzyme
thdF	BSU41020	No	Yes		1XZP	T. maritima	GTP-binding protein, putative tRNA modification GTPase
truA	BSU01480	No	Yes	5.4.99.12	1VS3	T. thermophilus	Pseudouridylate synthase I, universally conserved protein
tsaE	BSU05910	No	Yes		1HTW	H. influenzae	P-loop ATPase
trmFO	BSU16130	No	Yes	2.1.1.74	3G5Q	T. thermophilus	tRNA:m(5)U-54 methyltransferase
miaA	BSU17330	No		2.5.1.8	2QGN	Bacillus halodurans	tRNA isopentenylpyrophosphate transferase
yaaJ	BSU00180	No			2B3J	S. aureus	tRNA-specific adenosine deaminase
ylyB	BSU15460	No	Yes	5.4.99.23	1V9F	E. coli	Similar to pseudouridylate synthase
Ribosome maturation/assembly
ydiD	BSU05930	No		2.3.1.128	2CNM	S. enterica	Similar to ribosomal protein alanine N-acetyltransferase
ylxS	BSU16590	No			1IB8	S. pneumoniae	Similar to 30S ribosomal subunit maturation protein
prp	BSU27950	No			4PEO	S. aureus	Maturation of L27
engA	BSU22840	Yes			2HJG	B. subtilis	GTPase, ribosome 50S subunit assembly
era	BSU25290	Yes	Yes		3R9W	A. aeolicus	GTP-binding protein
obg	BSU27920	Yes	Yes	3.6.5.-	1LNZ	B. subtilis	GTP-binding protein
rbgA	BSU16050	Yes	Yes		1PUJ	B. subtilis	Assembly of the 50S subunit of the ribosome
yqeH	BSU25670	Yes			3H2Y	B. anthracis	Assembly/stability of the 30S subunit of the ribosome, assembly of the 70S ribosome
ysxC	BSU28190	Yes	Yes		1SVI	B. subtilis	Assembly of the 50S subunit of the ribosome
Translation factors
efp	BSU24450	No	Yes		1YBY	Clostridium thermocellum	Elongation factor P
frr	BSU16520	Yes	Yes		4GFQ	B. anthracis	Ribosome recycling factor
fusA	BSU01120	Yes	Yes		2XEX	S. aureus	Elongation factor G
infA	BSU01390	Yes	Yes		4QL5	S. pneumoniae	Translation initiation factor IF-1
infB	BSU16630	Yes	Yes		1ZO1	E. coli	Translation initiation factor IF-2
infC	BSU28870	Yes	Yes		1TIG	G. stearothermophilus	Translation initiation factor IF-3
prfA	BSU37010	Yes	Yes		1ZBT	Streptococcus mutans	Peptide chain release factor 1
prfB	BSU35290	Yes			1MI6	E. coli	Peptide chain release factor 2
tsf	BSU16500	Yes	Yes		1EFU	E. coli	Elongation factor Ts
tufA	BSU01130	Yes	Yes	3.6.5.3	4R71	E. coli	Elongation factor Tu
lepA	BSU25510	No	Yes		4QJT	T. thermophilus	Elongation factor 4
Translation/others
map	BSU01380	Yes	Yes	3.4.11.18	1O0X	T. maritima	Methionine aminopeptidase
ywkE	BSU37000	No	Yes	2.1.1.297	2B3T	E. coli	Similar to N⁵-glutamine methyltransferase that modifies peptide release factors
ybxF	BSU01090	No			3V7E	B. subtilis	Similar to ribosomal protein L7 family
spoVC	BSU00530	Yes	Yes	3.1.1.29	4QT4	S. pyogenes	Putative peptidyl-tRNA hydrolase
ssrA	BSU_MISC_RNA_55	No			1P6V	A. aeolicus	tmRNA
smpB	BSU33600	No	Yes		1P6V	A. aeolicus	tmRNA-binding protein
Protein secretion
scr	BSU_misc_RNA_2	Yes			4UE5	B. subtilis	Signal recognition particle RNA
ffh	BSU15980	Yes	Yes		4UE5	B. subtilis	Signal recognition particle component
ftsY	BSU15950	No	Yes		2XXA	E. coli	Signal recognition particle
yidC2	BSU23890	No			3WO6	B. halodurans	Sec-independent membrane protein translocase
secA	BSU35300	Yes	Yes		3DL8	B. subtilis	Preprotein translocase subunit (ATPase)
secE	BSU01000	Yes			3DL8	B. subtilis	Preprotein translocase subunit
secY	BSU01360	Yes	Yes		3DL8	B. subtilis	Preprotein translocase subunit, universally conserved protein
secG	BSU33630	No			3DL8	B. subtilis	Preprotein translocase subunit
sipS	BSU23310	No			4NV4	B. anthracis	Signal peptidase I
prsA	BSU09950	Yes		5.2.1.8	4WO7	B. subtilis	Protein secretion (posttranslocation molecular chaperone)
csaA	BSU19040	No			2NZH	B. subtilis	Molecular chaperone involved in protein secretion
lgt	BSU34990	No		2.4.99.-	5AZB	E. coli	Prolipoprotein diacylglyceryl transferase
lspA	BSU15450	No			5DIR	P. aeruginosa	Signal peptidase II
Proteolysis/quality control/chaperones
htrB	BSU33000	No			3QO6	Arabidopsis thaliana	Serine protease
groES	BSU06020	Yes			1WE3	T. thermophilus	Chaperonin, universally conserved protein
groEL	BSU06030	Yes			1WE3	T. thermophilus	Chaperonin
dnaJ	BSU25460	No	Yes		3LZ8	K. pneumoniae	Activation of DnaK
dnaK	BSU25470	No	Yes		2V7Y	G. kaustophilus	Molecular chaperone
grpE	BSU25480	No			4ANI	G. kaustophilus	Activation of DnaK
tig	BSU28230	No			2MLX	E. coli	Trigger factor (prolyl isomerase)
Metabolism
Central carbon metabolism
Glycolysis
ptsG	BSU13890	No	Yes	2.7.1.69			PTS glucose permease, EIICBA(Glc)
ptsH	BSU13900	No	Yes	2.7.11.-	2FEP	B. subtilis	HPr, general component of the PTS
ptsI	BSU13910	No	Yes	2.7.3.9	2WQD	S. aureus	Enzyme I, general component of the PTS
pgi	BSU31350	No	Yes	5.3.1.9	3IFS	B. anthracis	Glucose-6-phosphate isomerase
pfkA	BSU29190	No	Yes	2.7.1.11	4A3S	B. subtilis	Phosphofructokinase
fbaA	BSU37120	No		4.1.2.13	4TO8	S. aureus	Fructose 1,6-bisphosphate aldolase
tpi	BSU33920	No	Yes	5.3.1.1	2BTM	G. stearothermophilus	Triose phosphate isomerase
gapA	BSU33940	Yes	Yes	1.2.1.12	1GD1	G. stearothermophilus	Glyceraldehyde-3-phosphate dehydrogenase
pgk	BSU33930	No	Yes	2.7.2.3	1PHP	G. stearothermophilus	Phosphoglycerate kinase, universally conserved protein
pgm	BSU33910	Yes	Yes	5.4.2.1	1EJJ	G. stearothermophilus	Phosphoglycerate mutase
eno	BSU33900	Yes	Yes	4.2.1.11	4A3R	B. subtilis	Enolase, universally conserved protein
pyk	BSU29180	No	Yes	2.7.1.40	2E28	G. stearothermophilus	Pyruvate kinase
pdhA	BSU14580	Yes	Yes	1.2.4.1	3DUF	G. stearothermophilus	Pyruvate dehydrogenase (E1 alpha subunit)
pdhB	BSU14590	No	Yes	1.2.4.1	3DUF	G. stearothermophilus	Pyruvate dehydrogenase (E1 beta subunit)
pdhC	BSU14600	No	Yes	2.3.1.12	3DUF	G. stearothermophilus	Pyruvate dehydrogenase (dihydrolipoamide acetyltransferase E2 subunit)
pdhD	BSU14610	No	Yes	1.8.1.4	1EBD	G. stearothermophilus	Dihydrolipoamide dehydrogenase E3 subunit of both pyruvate and 2-oxoglutarate dehydrogenase complexes
Transhydrogenation cycle
ytsJ	BSU29220	No		1.1.1.38	2A9F	S. pyogenes	Malic enzyme
malS	BSU29880	No		1.1.1.38	1LLQ	Ascaris suum	Malate dehydrogenase (decarboxylating)
Pentose phosphate pathway
ykgB	BSU13010	No		3.1.1.31	3HFQ	Lactobacillus plantarum	6-Phosphogluconolactonase
rpe	BSU15790	No	Yes	5.1.3.1	1TQJ	Synechocystis sp.	Ribulose 5-phosphate 3-epimerase
tkt	BSU17890	No	Yes	2.2.1.1	3HYL	B. anthracis	Transketolase
zwf	BSU23850	No		1.1.1.49	1DPG	Leuconostoc mesenteroides	Glucose-6-phosphate dehydrogenase
gndA	BSU23860	No		1.1.1.44	2W8Z	G. stearothermophilus	NADP-dependent phosphogluconate dehydrogenase
ywlF	BSU36920	No	Yes		3HE8	C. thermocellum	Ribose-5-phosphate isomerase
ywjH	BSU37110	No		2.2.1.2	3R8R	B. subtilis	Transaldolase
Recycling of acetate
acsA	BSU29680	No		6.2.1.1	2P2F	S. enterica	Acetyl-CoA synthetase
Respiration/energy
ndh	BSU12290	No			4NWZ	Caldalkalibacillus thermarum	NADH dehydrogenase
Cytochrome aa₃
qoxD	BSU38140	No					Cytochrome aa₃ quinol oxidase (subunit IV)
qoxC	BSU38150	No			1FFT	E. coli	Cytochrome aa₃ quinol oxidase (subunit III)
qoxB	BSU38160	No			1FFT	E. coli	Cytochrome aa₃ quinol oxidase (subunit I)
qoxA	BSU38170	No			1FFT	E. coli	Cytochrome aa₃ quinol oxidase (subunit II)
Cytochrome maturation
resC	BSU23130	Yes					Part of heme translocase, required for cytochrome c synthesis
resB	BSU23140	Yes					Part of heme translocase, required for cytochrome c synthesis
ATPase
atpC	BSU36800	No			2E5Y	Bacillus sp.	ATP synthase, F₁ (subunit epsilon)
atpD	BSU36810	No	Yes	3.6.3.14	1SKY	Bacillus sp.	ATP synthase, F₁ (subunit beta)
atpG	BSU36820	No	Yes		4XD7	G. kaustophilus	ATP synthase, F₁ (subunit gamma)
atpA	BSU36830	No	Yes	3.6.3.14	1SKY	Bacillus sp.	ATP synthase, F₁ (subunit alpha)
atpH	BSU36840	No					ATP synthase, F₁ (subunit delta)
atpF	BSU36850	No	Yes				ATP synthase, F_o (subunit b)
atpE	BSU36860	No			1WU0	Bacillus sp.	ATP synthase, F_o (subunit c)
atpB	BSU36870	No			1C17	E. coli	ATP synthase, F_o (subunit a)
atpI	BSU36880	No					ATP synthase (subunit i)
Amino acids
Asp, Glu
gltT	BSU10220	No			3V8F	P. horikoshii	Major H⁺/Na⁺-glutamate symport protein
Arg
rocE	BSU40330	No			3LRB	E. coli	Amino acid permease
Pro
putP	BSU03220	No			2XQ2	Vibrio parahaemolyticus	High-affinity proline permease
Trp
trpP	BSU10010	No					S protein of tryptophan ECF transporter
Met
metQ	BSU32730	No			4GOT	B. subtilis	Methionine ABC transporter (binding lipoprotein)
metP	BSU32740	No			3DHW	E. coli	Methionine ABC transporter, permease
metN	BSU32750	No			3DHW	E. coli	Methionine ABC transporter (ATP-binding protein)
His
hutM	BSU39390	No					Histidine permease
Cys
tcyP	BSU09130	No	Yes		3KBC	P. horikoshii	Cystine transporter
Gly
glyA	BSU36900	Yes	Yes	2.1.2.1	1KKJ	G. stearothermophilus	Serine hydroxymethyltransferase
Ile, Val, Thr
bcaP	BSU09460	No					Branched-chain amino acid transporter
Lys
yvsH	BSU33330	No			3LRB	E. coli	Putative lysine transporter
Chorismate for aromatic amino acids, menaquinone, and folate
aroA	BSU29750	No		2.5.1.54	3NVT	L. monocytogenes	3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase/chorismate mutase isozyme 3
aroB	BSU22700	No		4.2.3.4	3CLH	Helicobacter pylori	3-Dehydroquinate synthase
aroC	BSU23080	No		4.2.1.10	1QFE	S. enterica serovar Typhi	3-Dehydroquinate dehydratase
aroD	BSU25660	No		1.1.1.25	2EGG	G. kaustophilus	Shikimate dehydrogenase
aroE	BSU22600	No		2.5.1.19	3RMT	B. halodurans	3-Phosphoshikimate 1-carboxyvinyltransferase
aroF	BSU22710	No		4.2.3.5	1Q1L	A. aeolicus	Chorismate synthase
aroK	BSU03150	No		2.7.1.71	2PT5	A. aeolicus	Shikimate kinase
Phe, Tyr
pheA	BSU27900	No		4.2.1.51	4LUB	S. mutans	Prephenate dehydratase
hisC	BSU22620	No		2.6.1.9	3FFH	Listeria innocua	Histidinol-phosphate aminotransferase/tyrosine and phenylalanine aminotransferase
aroH	BSU22690	No			1COM	B. subtilis	Chorismate mutase (isozymes 1 and 2)
tyrA	BSU22610	No		1.3.1.12	3DZB	Streptococcus thermophilus	Prephenate dehydrogenase
Asn
asnB	BSU30540	No		6.3.5.4	1CT9	E. coli	Asparagine synthase (glutamine hydrolyzing)
Ala, Ser
alaT	BSU31400	No		2.6.1.-	1DJU	P. horikoshii	Alanine aminotransferase
serA	BSU23070	No		1.1.1.95	1YGY	Mycobacterium tuberculosis	Phosphoglycerate dehydrogenase
serC	BSU10020	No		2.6.1.52	1W23	Bacillus alcalophilus	3-Phosphoserine aminotransferase
ywtE	BSU35850	No			1NRW	B. subtilis	Putative phosphatase
Leu
yvbW	BSU34010	No			3GI9	Methanocaldococcus jannaschii	Putative leucine permease
Gln
glnA	BSU17460	No		6.3.1.2	4S0R	B. subtilis	Glutamine synthetase
Nucleotides/phosphate
PRPP
prs	BSU00510	Yes	Yes	2.7.6.1	1DKR	B. subtilis	Phosphoribosylpyrophosphate synthetase, universally conserved protein
Pyrimidine biosynthesis
pyrAA	BSU15510	No		6.3.5.5	1JDB	E. coli	Carbamoyl-phosphate synthetase (glutaminase subunit)
pyrAB	BSU15520	No		6.3.5.5	1JDB	E. coli	Carbamoyl-phosphate synthetase (catalytic subunit)
pyrB	BSU15490	No		2.1.3.2	3R7D	B. subtilis	Aspartate carbamoyltransferase
pyrC	BSU15500	No		3.5.2.3	3MPG	B. anthracis	Dihydro-orotase
pyrD	BSU15540	No		1.3.3.1	1EP1	Lactococcus lactis	Dihydro-orotic acid dehydrogenase (catalytic subunit)
pyrE	BSU15560	No		2.4.2.10	3M3H	B. anthracis	Orotate phosphoribosyltransferase
pyrF	BSU15550	No		4.1.1.23	1DBT	B. subtilis	Orotidine 5′-phosphate decarboxylase
cmk	BSU22890	Yes	Yes	2.7.4.14	1Q3T	S. pneumoniae	Cytidylate kinase (CMP, dCMP)
pyrG	BSU37150	Yes	Yes	6.3.4.2	1S1M	E. coli	CTP synthase (NH₃, glutamine)
yncF	BSU17660	No			2XCD	B. subtilis	dUTPase
thyB	BSU21820	No		2.1.1.45	3IX6	Brucella melitensis	Thymidylate synthase B
tmk	BSU00280	Yes	Yes	2.7.4.9	2CCJ	S. aureus	Thymidylate kinase
Purine biosynthesis
purF	BSU06490	No		2.4.2.14	1GPH	B. subtilis	Glutamine phosphoribosyldiphosphate amidotransferase
purD	BSU06530	No		6.3.4.13	2XD4	B. subtilis	Phosphoribosylglycinamide synthetase
purN	BSU06510	No		2.1.2.2	3AV3	G. kaustophilus	Phosphoribosylglycinamide formyltransferase
purS	BSU06460	No			1TWJ	B. subtilis	Phosphoribosylformylglycinamidine synthase
purQ	BSU06470	No		6.3.5.3	3D54	T. maritima	Phosphoribosylformylglycinamidine synthase
purL	BSU06480	No		6.3.5.3	3VIU	T. thermophilus	Phosphoribosylformylglycinamidine synthase
purM	BSU06500	No		6.3.3.1	2BTU	B. anthracis	Phosphoribosylaminoimidazole synthetase
purE	BSU06420	No		4.1.1.21	1XMP	B. anthracis	Phosphoribosylaminoimidazole carboxylase (ATP dependent)
purK	BSU06430	No		4.1.1.21	4DLK	B. anthracis	Phosphoribosylaminoimidazole carboxylase (ATP dependent)
purC	BSU06450	No		6.3.2.6	2YWV	G. kaustophilus	Phosphoribosylaminoimidazole succinocarboxamide synthase
purB	BSU06440	No		4.3.2.2	1F1O	B. subtilis	Adenylsuccinate lyase
purH	BSU06520	No		2.1.2.3	3ZZM	M. tuberculosis	Phosphoribosylaminoimidazole carboxamide formyltransferase
guaB	BSU00090	Yes		1.1.1.205	3TSB	B. anthracis	IMP dehydrogenase
guaA	BSU06360	No		6.3.5.2	1GPM	E. coli	GMP synthase (glutamine hydrolyzing)
gmk	BSU15680	Yes	Yes	2.7.4.8	3TAU	L. monocytogenes	Guanylate kinase (GMP:dATP, dGMP:ATP)
purA	BSU40420	No		6.3.4.4	4M0G	B. anthracis	Adenylosuccinate synthetase
adk	BSU01370	Yes	Yes	2.7.4.3	1P3J	B. subtilis	Adenylate kinase
Pyrimidine/purine biosynthesis
nrdE	BSU17380	Yes	Yes		1PEM	S. enterica	Ribonucleoside diphosphate reductase (major subunit)
nrdF	BSU17390	Yes	Yes		4DR0	B. subtilis	Ribonucleoside diphosphate reductase (major subunit)
nrdI	BSU17370	Yes			1RLJ	B. subtilis	Ribonucleoside diphosphate reductase
ndk	BSU22730	No		2.7.4.6	2VU5	B. anthracis	Nucleoside diphosphate kinase
hprT	BSU00680	Yes	Yes	2.4.2.8	3H83	B. anthracis	Hypoxanthine phosphoribosyltransferase
Phosphate
pit	BSU12840	No					Low-affinity inorganic phosphate transporter
Lipids
Malonyl-CoA synthesis
accC	BSU24340	Yes		6.3.4.14	2VPQ	S. aureus	Acetyl-CoA carboxylase (biotin carboxylase subunit)
accB	BSU24350	Yes		6.4.1.2	4HR7	E. coli	Acetyl-CoA carboxylase (biotin carboxyl carrier subunit)
accA	BSU29200	Yes		6.4.1.2	2F9I	S. aureus	Acetyl-CoA carboxylase (alpha subunit)
accD	BSU29210	Yes		6.4.1.2	2F9I	S. aureus	Acetyl-CoA carboxylase (beta subunit)
birA	BSU22440	Yes		6.3.4.15	3RIR	S. aureus	Biotin protein ligase
Acyl carrier
acpS	BSU04620	Yes		2.7.8.7	1F80	B. subtilis	Acyl carrier protein synthase, 4′-phosphopantetheine transferase
acpA	BSU15920	Yes			1F80	B. subtilis	Acyl carrier protein
Aceto-acyl-Acp synthesis
fabD	BSU15900	Yes		2.3.1.39	3QAT	Bartonella henselae	Malonyl-CoA—acyl carrier protein transacylase
fabHA	BSU11330	No		2.3.1.180	1ZOW	S. aureus	Beta-ketoacyl–acyl carrier protein synthase III
β-Ketoacyl-Acp chain elongation
fabG	BSU15910	Yes		1.1.1.100	2UVD	B. anthracis	Beta-ketoacyl–acyl carrier protein reductase
fabF	BSU11340	Yes		2.3.1.179	4LS5	B. subtilis	Beta-ketoacyl–acyl carrier protein synthase II
fabI	BSU11720	No		1.3.1.9	3OIF	B. subtilis	Enoyl-acyl carrier protein reductase
ywpB	BSU36370	Yes			1U1Z	P. aeruginosa	β-Hydroxyacyl (acyl carrier protein) dehydratase
Phosphatidic acid synthesis
plsC	BSU09540	Yes		2.3.1.51			Acyl-ACP:1-acylglycerolphosphate acyltransferase
plsX	BSU15890	Yes	Yes		1VI1	B. subtilis	Acyl-ACP:phosphate acyltransferase
plsY	BSU18070	Yes	Yes				Acylphosphate:glycerol-phosphate acyltransferase
gpsA	BSU22830	Yes		1.1.1.94	1Z82	T. maritima	Glycerol-3-phosphate dehydrogenase (NAD)
Phosphatidylglycerol phosphate synthesis
cdsA	BSU16540	Yes	Yes	2.7.7.41	4Q2G	T. maritima	Phosphatidate cytidylyltransferase
pgsA	BSU16920	Yes	Yes	2.7.8.5			Phosphatidylglycerophosphate synthase
Cofactors
ECF transporter (general component) for riboflavin, biotin, thiamine, tryptophan
ybxA	BSU01450	No	Yes		4HUQ	Lactobacillus brevis	ATP-binding A1 component of ECF transporters
ybaE	BSU01460	No	Yes		4HUQ	L. brevis	ATP-binding A2 component of ECF transporters
ybaF	BSU01470	No	Yes		4HUQ	L. brevis	Transmembrane T component of ECF transporters
NAD
nadD	BSU25640	Yes		2.7.7.18	1KAM	B. subtilis	Nicotinamide-nucleotide adenylyltransferase
nadE	BSU03130	Yes	Yes	6.3.1.5	1NSY	B. subtilis	NH₃-dependent NAD⁺ synthetase
nadF	BSU11610	Yes	Yes	2.7.1.23	2I1W	L. monocytogenes	NAD kinase
niaP	BSU02950	No			4J05	Piriformospora indica	Nicotinate transporter
pncB	BSU31750	Yes		2.4.2.11	2F7F	E. faecalis	Putative nicotinate phosphoribosyltransferase
Riboflavin/FAD
ribC	BSU16670	Yes		2.7.1.26	3OP1	S. pneumoniae	Riboflavin kinase/FAD synthase
ribU	BSU23050	No			3P5N	S. aureus	Riboflavin ECF transporter, S protein
Pyridoxal phosphate
pdxS	BSU00110	No			2NV2	B. subtilis	Pyridoxal-5′-phosphate synthase (synthase domain)
pdxT	BSU00120	No			2NV2	B. subtilis	Pyridoxal-5′-phosphate synthase (glutaminase domain)
Biotin
yhfU	BSU10370	No			4DVE	L. lactis	S protein of biotin ECF transporter
Thiamine, TPP
yloS	BSU15800	No		2.7.6.2	3LM8	B. subtilis	Thiamine pyrophosphokinase
thiT	BSU30990	No			4MES	L. lactis	S protein of thiamine ECF transporter
Lipoate
gcvH	BSU32800	No			3IFT	M. tuberculosis	Glycine cleavage system protein H, 2-oxo acid dehydrogenase
lipM	BSU24530	No			3A7A	E. coli	Octanoyltransferase
lipL	BSU37640	No			2P5I	B. halodurans	GcvH:E2 amidotransferase
lipA	BSU32330	Yes		2.8.1.8	4U0O	T. elongatus	Lipoic acid synthase
CoA
ykpB	BSU14440	No			3HN2	Geobacter metallireducens	Putative ketopantoate reductase
panD	BSU22410	No		4.1.1.11	2C45	M. tuberculosis	Aspartate 1-decarboxylase
panC	BSU22420	No		6.3.2.1	2X3F	S. aureus	Pantothenate synthase
panB	BSU22430	No		2.1.2.11	1M3U	E. coli	3-Methyl-2-oxobutanoate hydroxymethyltransferase
ybgE	BSU02390	No		2.6.1.42	3HT5	M. tuberculosis	Branched-chain amino acid aminotransferase
coaA	BSU23760	No		2.7.1.33	4F7W	K. pneumoniae	Probable pantothenate kinase
yloI	BSU15700	No		4.1.1.36	1U7U	E. coli	Coenzyme A biosynthesis bifunctional protein CoaBC
ylbI	BSU15020	No		2.7.7.3	1O6B	B. subtilis	Pantetheine-phosphate adenylyltransferase
ytaG	BSU29060	Yes		2.7.1.24	4TTP	Legionella pneumophila	Dephospho-CoA kinase
SAM
metK	BSU30550	Yes	Yes	2.5.1.6	1FUG	E. coli	S-Adenosylmethionine synthetase
Folate
folE	BSU22780	No		3.5.4.16	4UQF	L. monocytogenes	GTP cyclohydrolase I
phoB	BSU05740	No		3.1.3.1	3A52	Shewanella sp.	Alkaline phosphatase A
folB	BSU00780	No		4.1.2.25	1RRI	S. aureus	Dihydroneopterin aldolase
folK	BSU00790	No		2.7.6.3	4CYU	S. aureus	2-Amino-4-hydroxy-6-hydroxymethyl-dihydropteridine diphosphokinase
sul	BSU00770	No		2.5.1.15	1TWS	B. anthracis	Dihydropteroate synthase
folC	BSU28080	No	Yes	6.3.2.17	1O5Z	T. maritima	Folyl-polyglutamate synthetase
dfrA	BSU21810	Yes		1.5.1.3	1ZDR	G. stearothermophilus	Dihydrofolate reductase
pabB	BSU00740	No		2.6.1.85	5CWA	M. tuberculosis	p-Aminobenzoate synthase (subunit A)
pabA	BSU00750	No		2.6.1.85	1I1Q	S. enterica serovar Typhimurium	p-Aminobenzoate synthase (subunit B)/anthranilate synthase (subunit II)
pabC	BSU00760	No		4.1.3.38	4WHX	Burkholderia pseudomallei	Aminodeoxychorismate lyase
gsaB	BSU08710	No		5.4.3.8	3L44	B. anthracis	Formate dehydrogenase
ykkE	BSU13110	No		3.5.1.10	3W7B	T. thermophilus	Formyltetrahydrofolate deformylase
yoaE	BSU18570	No					Formate dehydrogenase
Heme biosynthesis
hemE	BSU10120	No		4.1.1.37	2INF	B. subtilis	Glutamate-1-semialdehyde aminotransferase
hemH	BSU10130	No			1C1H	B. subtilis	Uroporphyrinogen decarboxylase (uroporphyrinogen III)
hemY	BSU10140	No		1.3.3.4	3I6D	B. subtilis	Ferrochelatase
ctaA	BSU14870	No					Protoporphyrinogen IX oxidase
ctaB	BSU14880	No					Heme A synthase
hemL	BSU28120	No		5.4.3.8	3BS8	B. subtilis	Heme O synthase (major enzyme)
hemB	BSU28130	No		4.2.1.24	1W5Q	P. aeruginosa	Glutamate-1-semialdehyde aminotransferase
hemD	BSU28140	No		4.2.1.75			Porphobilinogen synthase
hemC	BSU28150	No		2.5.1.61	4MLQ	Bacillus megaterium	Uroporphyrinogen III synthase
hemX	BSU28160	No					Hydroxymethylbilane synthase
hemA	BSU28170	No		1.2.1.70	4N7R	A. thaliana	Glutamyl-tRNA reductase
hemQ	BSU37670	No			1T0T	G. stearothermophilus	Heme-binding protein
Menaquinone
menA	BSU38490	Yes					Probable 1,4-dihydroxy-2-naphthoate octaprenyltransferase
menH	BSU22750	No			4OBW	Saccharomyces cerevisiae	Menaquinone biosynthesis methyltransferase
menC	BSU30780	Yes		4.2.1.113	1WUE	E. faecalis	O-Succinylbenzoate-CoA synthase
menE	BSU30790	Yes		6.2.1.26	5BUQ	B. subtilis	O-Succinylbenzoate-CoA ligase
menB	BSU30800	Yes		4.1.3.36	2IEX	G. kaustophilus	Naphthoate synthase
menD	BSU30820	Yes		2.2.1.9	2X7J	B. subtilis	2-Succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase/2-oxoglutarate decarboxylase
ytxM	BSU30810	No			2XMZ	S. aureus	Similar to prolyl aminopeptidase
menF	BSU30830	No		5.4.4.2	3HWO	E. coli	Menaquinone-specific isochorismate synthase
Metals and iron-sulfur clusters
Sodium export
mrpA	BSU31600	Yes			4HE8	T. thermophilus	Na⁺/H⁺ antiporter subunit
mrpB	BSU31610	Yes					Na⁺/H⁺ antiporter subunit
mrpC	BSU31620	Yes					Na⁺/H⁺ antiporter subunit
mrpD	BSU31630	Yes			4HE8	T. thermophilus	Na⁺/H⁺ antiporter subunit
mrpE	BSU31640	No					Na⁺/H⁺ antiporter subunit
mrpF	BSU31650	Yes					Na⁺/H⁺ antiporter subunit
mrpG	BSU31660	No					Na⁺/H⁺ antiporter subunit
Potassium
ktrD	BSU13500	No	Yes		4J7C	B. subtilis	Potassium transporter KtrCD
ktrC	BSU14510	No	Yes		4J90	B. subtilis	Potassium transporter KtrCD
Iron
efeO	BSU38270	No			3AT7	Sphingomonas sp.	Lipoprotein, elemental iron uptake system (binding protein)
efeU	BSU38280	No					Elemental iron uptake system (permease)
yfmF	BSU07490	No			4G1U	Yersinia pestis	Iron/citrate ABC transporter (ATP-binding protein)
yfmE	BSU07500	No			4G1U	Y. pestis	Iron/citrate ABC transporter (permease)
yfmD	BSU07510	No			4G1U	Y. pestis	Iron/citrate ABC transporter (permease)
yfmC	BSU07520	No			3EIW	S. aureus	Iron/citrate ABC transporter (binding protein)
yhfQ	BSU10330	No			3EIW	S. aureus	Iron/citrate ABC transporter (solute-binding protein)
Magnesium
mgtE	BSU13300	No			2YVX	T. thermophilus	Primary magnesium transporter
mntH	BSU04360	No			4WGV	Staphylococcus capitis	Manganese transporter (proton symport)
Zinc
znuA	BSU02850	No			2O1E	B. subtilis	ABC transporter for zinc (binding protein)
znuC	BSU02860	No			4YMS	Caldanaerobacter subterraneus	ABC transporter for zinc (ATP-binding protein)
znuB	BSU02870	No					ABC transporter for zinc (permease)
Copper
ycnJ	BSU03950	No					Copper transporter
Fe-S cluster
sufB	BSU32670	Yes			5AWF	E. coli	Synthesis of Fe-S clusters
sufU	BSU32680	Yes	Yes		1XJS	B. subtilis	Iron-sulfur cluster scaffold protein
sufD	BSU32700	Yes			5AWF	E. coli	Synthesis of Fe-S clusters
sufS	BSU32690	Yes	Yes	2.8.1.7	1T3I	Synechocystis sp.	Cysteine desulfurase
sufC	BSU32710	Yes			2D2E	T. thermophilus	ABC transporter (ATP-binding protein)
fra	BSU05750	No			2OC6	B. subtilis	Frataxin-like protein
yutI	BSU32220	No			1XHJ	Staphylococcus epidermidis	Putative iron-sulfur scaffold protein
Cell division
Cell wall synthesis
Synthesis of d-glutamate
racE	BSU28390	Yes		5.1.1.3	1ZUW	B. subtilis	Glutamate racemase
Synthesis of d-Ala-d-Ala
alr	BSU04640	Yes		5.1.1.1	3ZM5	S. pneumoniae	Alanine racemase
ddl	BSU04560	Yes		6.3.2.4	2I80	S. aureus	d-Alanine-d-alanine ligase
Synthesis of m-diaminopimelate
dapG	BSU16760	No		2.7.2.4	1SFT	G. stearothermophilus	Aspartokinase I (alpha and beta subunits)
asd	BSU16750	Yes		1.2.1.11	2GYY	S. pneumoniae	Aspartate-semialdehyde dehydrogenase
dapA	BSU16770	Yes		4.2.1.52	1XKY	B. anthracis	Dihydrodipicolinate synthase
dapB	BSU22490	Yes		1.3.1.26	5EER	Corynebacterium glutamicum	Dihydrodipicolinate reductase (NADPH)
ykuQ	BSU14180	Yes		2.3.1.89	3R8Y	B. anthracis	Similar to tetrahydrodipicolinate succinylase
patA	BSU14000	Yes			1GDE	P. horikoshii	Aminotransferase
dapI	BSU14190	Yes		3.5.1.47	1YSJ	B. subtilis	N-Acetyl-diaminopimelate deacetylase
dapF	BSU32170	Yes		5.1.1.7	2OTN	B. anthracis	Diaminopimelate epimerase
Isoprenoid biosynthesis
dxs	BSU24270	Yes		2.2.1.7	2O1S	E. coli	1-Deoxyxylulose-5-phosphate synthase
ispC	BSU16550	Yes		1.1.1.267	1R0K	Zymomonas mobilis	1-Deoxy-d-xylulose-5-phosphate reductoisomerase
ispD	BSU00900	Yes		2.7.7.60	2YC3	A. thaliana	2-C-Methyl-d-erythritol 4-phosphate cytidylyltransferase
ispE	BSU00460	Yes		2.7.1.148	3PYD	M. tuberculosis	4-Diphosphocytidyl-2-C-methyl-d-erythritol kinase
ispF	BSU00910	Yes		4.6.1.12	3GHZ	S. enterica serovar Typhimurium	2-C-Methyl-d-erythritol-2,4-cyclodiphosphate synthase
ispG	BSU25070	Yes			3NOY	A. aeolicus	Similar to peptidoglycan acetylation, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase
ispH	BSU25160	Yes			4N7B	Plasmodium falciparum	(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase
fni	BSU22870	No		5.3.3.2	1P0K	B. subtilis	Isopentenyl diphosphate isomerase
Undecaprenyl phosphate biosynthesis
yqiD	BSU24280	No		2.5.1.10	1RTR	S. aureus	Geranyltransferase
uppS	BSU16530	Yes		2.5.1.31	1F75	Micrococcus luteus	Probable undecaprenyl pyrophosphate synthetase
bcrC	BSU36530	No					Undecaprenyl pyrophosphate phosphatase
hepT	BSU22740	Yes		2.5.1.30	3AQB	M. luteus	Heptaprenyl diphosphate synthase component II
hepS	BSU22760	Yes		2.5.1.30			Heptaprenyl diphosphate synthase component I
Peptidoglycan biosynthesis
glmS	BSU01780	Yes		2.6.1.16	4AMV	E. coli	Glutamine:fructose-6-phosphate transaminase
glmM	BSU01770	Yes		5.4.2.10	3PDK	B. anthracis	Phosphoglucosamine mutase
gcaD	BSU00500	Yes		2.7.7.23	4AAW	S. pneumoniae	UDP-N-acetylglucosamine pyrophosphorylase
murAA	BSU36760	Yes		2.5.1.7	3SG1	B. anthracis	UDP-N-acetylglucosamine 1-carboxyvinyltransferase
murB	BSU15230	Yes		1.1.1.158	4PYT	Unidentified	UDP-N-acetylenolpyruvoylglucosamine reductase
murC	BSU29790	Yes		6.3.2.8	1GQQ	H. influenzae	UDP-N-acetylmuramoyl-l-alanine synthetase
murD	BSU15200	Yes		6.3.2.9	3LK7	Streptococcus agalactiae	UDP-N-acetylmuramoyl-l-alanyl-d-glutamate synthetase
murE	BSU15180	Yes		6.3.2.13	4C13	S. aureus	UDP-N-acetylmuramoyl-l-alanyl-d-glutamyl-meso-2,6-diaminopimelate synthetase
murF	BSU04570	Yes		6.3.2.10	1GG4	E. coli	UDP-N-acetylmuramoyl-l-alanyl-d-glutamyl-meso-2,6-diaminopimeloyl-d-alanyl-d-alanine synthetase
mraY	BSU15190	Yes		2.7.8.13	4J72	A. aeolicus	Phospho-N-acetylmuramoyl-pentapeptide transferase (meso-2,6-diaminopimelate)
murG	BSU15220	Yes		2.4.1.227	1F0K	E. coli	UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenol N-acetylglucosamine transferase
murJ	BSU30050	No					Lipid II flippase
Peptidoglycan polymerization
ponA	BSU22320	No			3DWK	S. aureus	Penicillin-binding protein 1A/1B
PG cross-links, cell separation
pbpB	BSU15160	Yes			1RP5	S. pneumoniae	Penicillin-binding protein 2B
pbpA	BSU25000	No			3VSK	S. aureus	Penicillin-binding protein 2A
lytE	BSU09420	No			4XCM	T. thermophilus	Cell wall hydrolase (major autolysin) for cell elongation and separation
lytF	BSU09370	No			4XCM	T. thermophilus	Gamma-d-glutamate-meso-diaminopimelate muropeptidase (major autolysin)
Wall teichoic acid
tagO	BSU35530	Yes			4J72	A. aeolicus	Undecaprenyl phosphate-GlcNAc-1-phosphate transferase
mnaA	BSU35660	Yes		5.1.3.14	4FKZ	B. subtilis	UDP-N-acetylglucosamine 2-epimerase
tagA	BSU35750	Yes		2.4.1.187			UDP-N-acetyl-d-mannosamine transferase
tagB	BSU35760	Yes			3L7I	S. epidermidis	Putative CDP-glycerol:glycerol phosphate glycerophosphotransferase
tagD	BSU35740	Yes		2.7.7.39	1COZ	B. subtilis	Glycerol-3-phosphate cytidylyltransferase
tagF	BSU35720	Yes		2.7.8.12	3L7I	S. epidermidis	CDP-glycerol:polyglycerol phosphate glycerophosphotransferase
tagH	BSU35700	Yes		3.6.3.40			ABC transporter for teichoic acid translocation (ATP-binding protein)
tagG	BSU35710	Yes					ABC transporter for teichoic acid translocation (permease)
tagU	BSU35650	No			3OWQ	L. innocua	Phosphotransferase, attachment of anionic polymers to peptidoglycan
Lipoteichoic acid
dgkB	BSU06720	Yes			2QV7	S. aureus	Diacylglycerol kinase
pgcA	BSU09310	No	Yes	5.4.2.2	2Z0F	T. thermophilus	Alpha-phosphoglucomutase
gtaB	BSU35670	No	Yes	2.7.7.9	2UX8	Sphingomonas elodea	UTP-glucose-1-phosphate uridylyltransferase
ltaS	BSU07710	No			2W8D	B. subtilis	Lipoteichoic acid synthase
ugtP	BSU21920	No					UDP-glucose diacylglycerol glucosyltransferase
Coordination
Divisome
divIC	BSU00620	Yes					Cell division initiation protein (septum formation)
ftsL	BSU15150	Yes					Cell division protein (septum formation)
divIB	BSU15240	Yes			1YR1	G. stearothermophilus	Cell division initiation protein (septum formation)
ftsZ	BSU15290	Yes			2VAM	B. subtilis	Cell division initiation protein (septum formation)
ftsW	BSU14850	Yes					Cell division protein
ezrA	BSU29610	No			4UXV	B. subtilis	Negative regulator of FtsZ ring formation
sepF	BSU15390	No			3ZIH	B. subtilis	Part of the divisome
gpsB	BSU22180	No			4UG3	B. subtilis	Removal of PBP1 from the cell pole after completion of cell pole maturation
yvcK	BSU34760	No			2HZB	B. halodurans	Correct localization of PBP1, essential for growth under gluconeogenic conditions
yvcL	BSU34750	No	Yes		3HYI	T. maritima	Involved in Z-ring assembly
Division site selection
divIVA	BSU15420	No			2WUJ	B. subtilis	Cell division initiation protein (septum placement)
minC	BSU28000	No			2M4I	B. subtilis	Cell division inhibitor (septum placement)
minD	BSU27990	No			4V03	A. aeolicus	Cell division inhibitor (septum placement)
noc	BSU40990	No			1VZ0	T. thermophilus	DNA-binding protein, spatial regulator of cell division to protect the nucleoid, coordination of chromosome segregation and cell division
minJ	BSU35220	No					Topological determinant of cell division
Elongasome
mreD	BSU28010	Yes					Cell shape-determining protein, associated with the MreB cytoskeleton
mreC	BSU28020	Yes			2J5U	L. monocytogenes	Cell shape-determining protein, associated with the MreB cytoskeleton
mreB	BSU28030	Yes			4CZE	Caulobacter vibrioides	Cell shape-determining protein
rodA	BSU38120	Yes					Control of cell shape and elongation
mreBH	BSU14470	No			1JCF	T. maritima	Cell shape-determining protein
rodZ	BSU16910	Maybe					Required for cell shape determination
Coordination of cell division and DNA replication
walJ	BSU40370	No			4P62	P. aeruginosa	Coordination of cell division and DNA replication
Signaling
walK	BSU40400	Yes			4I5S	S. mutans	Two-component sensor kinase
walR	BSU40410	Yes			2ZWM	B. subtilis	Two-component response regulator
cdaA	BSU01750	No		2.7.7.85	4RV7	L. monocytogenes	Diadenylate cyclase
gdpP	BSU40510	No					c-di-AMP-specific phosphodiesterase
Integrity of the cell
Protection
ytbE	BSU29050	Yes			3B3D	B. subtilis	Putative aldo/keto reductase
katA	BSU08820	No			1SI8	E. faecalis	Vegetative catalase 1
sodA	BSU25020	No			2RCV	B. subtilis	Superoxide dismutase
ahpC	BSU40090	No			1WE0	Amphibacillus xylanus	Alkyl hydroperoxide reductase (small subunit)
ahpF	BSU40100	No			4O5Q	E. coli	Alkyl hydroperoxide reductase (large subunit)/NADH dehydrogenase
trxA	BSU28500	Yes	Yes		2GZY	B. subtilis	Antioxidative action by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange
yumC	BSU32110	Yes		1.18.1.2	3LZW	B. subtilis	Ferredoxin-NAD(P)⁺ oxidoreductase
trxB	BSU34790	Yes	Yes	1.8.1.9	4GCM	S. aureus	Thioredoxin reductase (NADPH)
Repair/genome integrity
hlpB	BSU10660	Yes					HNH nuclease-like protein, rescues AddA recombination intermediates
mutY	BSU08630	No			5DPK	G. stearothermophilus	A/G-specific adenine glycosylase
polY1	BSU23870	No		2.7.7.7	4IRC	E. coli	Translesion synthesis DNA polymerase Y1
mutM	BSU29080	No		3.2.2.23	1L1T	G. stearothermophilus	Formamidopyrimidine-DNA glycosidase
mfd	BSU00550	No			2EYQ	E. coli	Transcription repair-coupling factor
recD2	BSU27480	No			3E1S	Deinococcus radiodurans	5′–3′ DNA helicase replication fork progression
rnhB	BSU16060	No			3O3G	T. maritima	RNase HII, endoribonuclease
recA	BSU16940	No			1UBC	Mycobacterium smegmatis	Homologous recombination and DNA repair
Other/unknown
ppaC	BSU40550	Yes		3.6.1.1	1WPM	B. subtilis	Inorganic pyrophosphatase
ylaN	BSU14840	Yes			2ODM	S. aureus	Unknown
yitI	BSU11000	No			2JDC	Bacillus licheniformis	Unknown
yitW	BSU11160	No			3LNO	B. anthracis	Unknown
yqhY	BSU24330	No					Unknown
ykwC	BSU13960	No			3WS7	Pyrobaculum calidifontis	Putative beta-hydroxy acid dehydrogenase
ylbN	BSU15070	No					Unknown
ypfD	BSU22880	No			4Q7J	E. coli	Similar to ribosomal protein S1
yugI	BSU31390	No			2K4K	B. subtilis	Similar to polyribonucleotide nucleotidyltransferase
floT	BSU31010	No					Similar to flotillin 1, orchestration of physiological processes in lipid microdomains
yyaF	BSU40920	No	Yes		1JAL	H. influenzae	GTP-binding protein/GTPase

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The BSU number is the locus tag in the context of the B. subtilis 168 genome (GenBank accession no. NC_000964).

The essentiality of a gene refers to wild-type B. subtilis 168. By definition, genes that cannot be deleted as individual genes under defined optimal growth conditions (Luria-Bertani broth with glucose at 37°C) are regarded as being essential.

Each gene of the MiniBacillus gene set was tested for the presence of a homolog in Mycoplasma mycoides JCVI-syn3.0 by using a BLASTP analysis.

The organism refers to the PDB accession number.

PRPP, phosphoribosyl pyrophosphate; TPP, thiamine PP_i; SAM, S-adenosylmethionine; PTS, phosphotransferase system; PBP1, penicillin-binding protein 1.

TABLE 3.

rRNAs and tRNAs

rRNA(s) and/or tRNA(s)
rrnO-16S trnO-Ala trnO-Ile rrnO-23S rrnO-5S
trnSL-Ser1
rrnA-16S trnA-Ile trnA-Ala rrnA-23S rrnA-5S
trnSL-Met1 trnSL-Glu1
rrnJ-16S rrnJ-23S rrnJ-5S trnJ-Ala trnJ-Arg trnJ-Gly trnJ-Leu1 trnJ-Leu2 trnJ-Lys trnJ-Pro trnJ-Thr trnJ-Val rrnW-16S rrnW-23S rrnW-5S
rrnI-16S rrnI-23S rrnI-5S trnI-Ala trnI-Arg trnI-Asn trnI-Gly trnI-Pro trnI-Thr rrnH-16S rrnH-23S rrnH-5S rrnG-16S rrnG-23S rrnG-5S
trnSL-Gln2 trnSL-Glu2 trnSL-Thr1 trnSL-Tyr1 trnSL-Val1
trnS-Asn trnS-Gln trnS-Glu trnS-Leu1 trnS-Leu2 trnS-Lys trnS-Ser
trnE-Arg trnE-Gly rrnE-16S rrnE-23S rrnE-5S trnE-Asp trnE-Met
rrnD-16S rrnD-23S rrnD-5S trnD-Asn trnD-Asp trnD-Cys trnD-Gln trnD-Glu trnD-Gly trnD-His trnD-Leu1 trnD-Leu2 trnD-Met trnD-Phe trnD-Ser trnD-Thr trnD-Trp trnD-Tyr trnD-Val
trnSL-Gly1
trnSL-Val2
trnSL-Arg1
trnSL-Gln1
trnSL-Arg2
rrnB-16S rrnB-23S rrnB-5S trnB-Ala trnB-Arg trnB-Asn trnB-Asp trnB-Glu trnB-Gly1 trnB-Gly2 trnB-His trnB-Ile2 trnB-Leu1 trnB-Leu2 trnB-Lys trnB-Met1 trnB-Met2 trnB-Met3 trnB-Phe trnB-Pro trnB-Ser1 trnB-Ser2 trnB-Thr trnB-Val
trnSL-Ala1
trnQ-Arg
trnY-Asp trnY-Glu trnY-Lys trnY-Phe

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DNA Replication and Chromosome Segregation/Maintenance

A set of 18 genes was selected as being important for DNA replication. Among these 18 genes are 15 essential genes, which are absolutely required for growth of B. subtilis. With the exception of the essential NAD-dependent DNA ligase LigA, all these essential proteins are members of one or both of the protein complexes that catalyze DNA replication, i.e., the primosome and the replisome (45, 46). In addition to these essential proteins, we have added the replication termination protein Rtp, the inhibitor of DnaA oligomerization YabA, and the DNA polymerase I PolA to the list. Rtp is required for the correct termination of DNA replication and the subsequent segregation of daughter chromosomes (47). YabA, on the other hand, controls replication initiation by inhibiting the oligomerization of the initiator protein DnaA (48). Finally, DNA polymerase I fulfills an essential function, the removal of RNA primers that initiate the synthesis of Okazaki fragments. This function can be taken over by the paralog YpcP, and one of the two proteins has to be present for viability of B. subtilis. While YpcP has only the 5′-to-3′ exonuclease domain required for the removal of RNA primers, polymerase I is also capable of filling the resulting gaps (49). This additional property as well as the genetic linkage with the mutM gene, which is necessary for DNA integrity (see below), suggested that PolA should be kept in the minimal genome. Interestingly, B. subtilis needs either PolA or YpcP, whereas the M. mycoides minimal strain JCVI-syn3.0 encodes both proteins.

After DNA replication, the daughter chromosomes have to be efficiently distributed to the daughter cells. Moreover, correct chromosome condensation is essential for all biological processes that involve DNA. Chromosome segregation and condensation are highly overlapping functions; i.e., several of the proteins mentioned below are involved in both activities (for a review, see reference 50). This functional group encompasses 13 proteins, 9 of which are essential. Seven of the corresponding essential genes, but none of the nonessential genes, are present in the genome of M. mycoides JCVI-syn3.0. After the initiation of DNA replication, the newly formed origin regions are bound and separated by the condensin protein complex. The condensin complex is composed of two subunits of the Smc protein and monomers of ScpA and ScpB (51, 52). The essential topoisomerase IV formed by ParC and ParE is required for both DNA condensation and segregation (53). For the separation and segregation of the chromosome terminus, the DNA translocases SpoIIIE and SftA and the site-specific recombinases CodV and RipX are necessary. In principle, SpoIIIE and SftA as well as CodV and RipX are paralogous proteins, and one protein might be sufficient for each function. However, as we are aiming for a well-growing strain that is not significantly compromised in its major cellular functions, and as synergistic activities of these proteins were recently shown (54 –56), we decided to keep all four genes. DNA topology is maintained by the interplay of gyrase and topoisomerase I activities, encoded by the essential gyrAB and topA genes, respectively (28, 57). Moreover, the histone-like protein HBsu nonspecifically binds the DNA and is involved in DNA packaging (58).

Transcription

Eight proteins, among them five essential proteins, are required for transcription. Importantly, this activity requires the RNA polymerase, which consists of the essential core subunits (RpoA, RpoB, and RpoC) and sigma factor A (SigA) for promoter binding and recognition. Moreover, we have included the RNA polymerase-interacting protein HelD and the nonessential delta subunit (RpoE). HelD binding stimulates transcription in an RpoE-dependent manner, suggesting that these two accessory proteins are important to allow rapid growth (59, 60). Interestingly, both proteins are absent from the rather slowly growing M. mycoides strain JCVI-syn3.0. Finally, GreA and the essential NusA protein are required for transcription elongation and termination, respectively (61, 62). In contrast, the transcription termination protein Rho does not affect the growth of B. subtilis in rich medium (63) and therefore has not been included in the list.

mRNA Folding and Degradation

Once the RNA has been formed by the RNA polymerase, it has to adopt a structure that is compatible with its function. The rRNAs and tRNAs adopt complex three-dimensional structures, whereas the mRNAs have to be unstructured to allow access to the ribosomes. Bacterial cells have to adapt very rapidly to changes of external conditions. A major component of this rapid adaptation is the rapid degradation of bacterial mRNAs, which have average half-lives in the minute range. RNA degradation is accomplished by a set of RNases (64). In total, six proteins are required for the proper folding and degradation of RNA. To keep RNA molecules unstructured, the cell possesses so-called RNA chaperones. Two of these RNA chaperones, CspB and CspD, belong to the most abundant proteins in B. subtilis during growth in LB medium with glucose at 37°C (43, 65). Moreover, cspD is also one of the most highly expressed genes of B. subtilis under more than 100 different conditions (39). These data suggest that these RNA chaperones should be included in the minimal cell. In contrast, we decided to exclude all DEAD-box RNA helicases since B. subtilis has no growth defect even in the absence of all four helicases at 37°C (66). Conflicting reports are available for RNases in B. subtilis. The endoribonuclease RNase Y and the 5′-to-3′ exonuclease RNase J1 have long been considered to be essential, whereas a recent report suggests that these proteins are dispensable (34, 67 –69). However, the strong expression of the corresponding genes suggests that they should be part of a minimal cell. The same is true for the 3′-to-5′ exoribonuclease PnpA, which also belongs to the most abundant proteins. Moreover, PnpA has also been implicated in DNA repair (70). Finally, we decided to include the nano-RNase NrnA, which degrades and thus recycles bi- and oligoribonucleotides (71). This enzyme is conserved in all groups of mollicutes despite their significant genome reduction (42). This strong conservation is suggestive of an important function and justifies the inclusion of this protein in the minimal cell. Moreover, RNases J1 and Y as well as the nano-RNase NrnA are also encoded by M. mycoides JCVI-syn3.0.

Translation

A large set of 133 proteins and 118 RNAs is required for translation. This includes aminoacyl-tRNA synthetases, ribosomal proteins, rRNAs, tRNAs, proteins involved in rRNA and tRNA modification and maturation, ribosome biogenesis factors, and translation factors.

Twenty-four proteins constitute the set of 20 aminoacyl-tRNA synthetases (72). With the exception of ThrS, the threonyl-tRNA synthetase, all of these enzymes are essential in B. subtilis. ThrS has a weakly expressed paralog, and one of these proteins is required for the viability of B. subtilis (36). Similarly, the enzyme for tyrosine, TyrS, has a paralog. However, this paralog is not expressed under the most common conditions, rendering TyrS essential (73). It should be noted that the aminoacyl-tRNA synthetases for glycine and phenylalanine are composed of two subunits, and the transamidosome for the formation of glutamine-tRNA is composed of the three subunits (GatA, GatB, and GatC) of the glutamyl-tRNA(Gln) amidotransferase, the glutamyl-tRNA synthetase GltX, and glutamine-specific tRNA (74). In the artificial minimal organism M. mycoides JCVI-syn3.0, there are no paralogs of the genes encoding the aminoacyl-tRNA synthetases for glycine and proline. It is tempting to speculate that these functions are fulfilled by some of the 149 unknown proteins encoded by this genome.

The B. subtilis ribosome consists of 53 proteins under standard conditions, among them 33 and 20 in the large and small subunits, respectively. Several of these proteins are nonessential; however, this has been tested only with single-gene inactivation (75). It is therefore conceivable that the simultaneous lack of multiple ribosomal proteins would have a significant impact on viability. In addition, B. subtilis encodes auxiliary ribosomal proteins that are synthesized only under stress conditions or that replace zinc-containing proteins during zinc limitation (76 –78). All these proteins were excluded from the list (Table 2). The genome of the artificial organism M. mycoides JCVI-syn3.0 lacks genes for the essential protein L30 (RpmD) as well as those for 10 nonessential ribosomal proteins, mainly of the large ribosomal subunit.

B. subtilis has 10 operons for rRNA. All these operons also contain genes for tRNAs. Moreover, several tRNAs are encoded by scattered genes. It is well established that the redundancy of rRNAs and tRNAs is an important determinant of the growth rate (79); therefore, we included all 30 and 86 genes encoding rRNAs and tRNAs, respectively, in the minimal genome. However, reduction of the copy numbers of rRNA and tRNA genes remains to be explored to achieve a robust minimal translation machinery.

To be functional, rRNAs and tRNAs have to be processed and modified. In the minimal gene set, we include 31 proteins and 1 RNA for rRNA and tRNA maturation/modification, respectively. Since the functional RNAs are usually expressed as parts of large operons, proper processing is the first important step in their maturation. It should be noted that RNases Y and J1 (see above) participate in the processing and maturation of these functional RNA molecules (80, 81). Moreover, RNases P, PH, and Z are involved in rRNA and tRNA processing. Of these, RNase P (composed of a protein and the catalytically active RNA component) and RNase Z are essential (82, 83). In addition, RimM and RbfA are important for ribonucleolytic 16S rRNA maturation (84, 85). rRNAs and tRNAs are subject to multiple and highly diverse modifications, including methylation, thiouridylation, and pseudouridylation. The modification of tRNAs is one of the functions that still needs substantial research effort. The proteins important for these activities have been derived from the SubtiWiki database and from a recent study on the minimal translation apparatus (33, 42). As shown in Table 2, there is a good match with the corresponding set of genes in M. mycoides JCVI-syn3.0 (10).

Nine proteins, including six essential proteins, are involved in ribosome maturation and assembly. The three nonessential proteins process or modify ribosomal proteins. The six essential proteins are all GTPases that participate in different aspects of ribosome assembly (86, 87). Four of the genes encoding these essential GTPases are also part of the genome of M. mycoides JCVI-syn3.0.

Eleven proteins function as translation factors in different steps of translation, i.e., initiation, elongation, peptide chain release, and ribosome recycling. Of these proteins, nine are essential. Of the nonessential proteins, Efp is important for the efficient translation of proteins containing multiple consecutive proline residues, among them three essential proteins (Fmt, TopA, and ValS) (see http://subtiwiki.uni-goettingen.de/wiki/index.php/Efp-dependent_proteins) (33, 88, 89). With the exception of peptide chain release factor 2 (PrfB), but including elongation factor P (Efp), all of these proteins are encoded by the genome of M. mycoides JCVI-syn3.0.

In addition, five proteins and one RNA are important for translation. These include the essential methionine aminopeptidase, which removes the N-terminal methionine from nascent proteins (90). In addition, transfer-messenger RNA (tmRNA) and its binding protein rescue stalled ribosomes, whereas the essential peptidyl-tRNA hydrolase SpoVC recycles tRNA molecules sequestered as peptidyl-tRNA as a result of premature dissociation from the ribosome (91 –93). The precise functions of YwkE and YbxF still have to be discovered. YbxF binds to turns in RNA molecules and is very strongly expressed under most conditions (33, 39, 94).

Protein Secretion

Several proteins of the minimal cell have to be targeted either to the membrane or for secretion into the medium. For this activity, the minimal cell needs a set of 12 proteins (5 of them essential) and 1 essential RNA for protein secretion (Fig. 2). The first component in cotranslational targeting of the membrane and secreted proteins is the universally conserved signal recognition particle composed of the essential RNA scr, the essential protein Ffh, and FtsY (95 –99). The next step in protein translocation is performed by the chaperone CsaA and by the translocation motor SecA, which provides the energy for the export of unfolded secretory precursor proteins through the channel formed by SecYEG (100 –102). Alternatively, YidC2 can translocate the preproteins through the membrane. In B. subtilis, YidC2 has a paralog, SpoIIIJ, and one of the two proteins is required for viability (36, 103, 104). We included YidC2 since this protein is also important for genetic competence (105) (see below). For secreted proteins, the signal peptide is then cleaved off by signal peptidase I (SipS) (106, 107). In the case of lipoproteins, the protein is diacylglyceryl modified by Lgt for attachment to the membrane, and signal peptide cleavage is subsequently performed by LspA (108 –110). Proper folding of extracellular proteins is assisted by the posttranslocation chaperone PrsA, a lipoprotein attached to the outer surface of the cytoplasmic membrane (111, 112). B. subtilis also possesses the TAT (twin arginine translocation) and type VII protein secretion systems. However, these systems are not essential in a minimal cell (113 –115). Interestingly, the reduced genome of M. mycoides JCVI-syn3.0 encodes only the signal recognition particle, the translocation motor SecA, and an incomplete Sec channel. Importantly, this minimal organism seems to lack enzymes for the export and membrane attachment of lipoproteins. It is tempting to speculate that these functions are encoded by some of the unknown genes, since lipoprotein-dependent metabolite uptake is crucial for Mycoplasma bacteria with their strongly reduced metabolism. This is the case not only for artificially genome-reduced bacteria but also for natural Mycoplasma species. In M. pneumoniae, lipoproteins account for about 10% of all encoded proteins (116, 117). Moreover, many of the 149 unknown genes in the minimal genome of M. mycoides JCVI-syn3.0 encode lipoproteins (10).

Intracellular Chaperones, Protein Quality Control, and Proteolysis

To be active, proteins have to be properly folded, and misfolded proteins need to be detected and then be refolded or degraded. These activities can be performed by a set of seven proteins, including two essential proteins. The universally conserved chaperone DnaK with its cochaperones DnaJ and GrpE, the trigger factor Tig, as well as the universally conserved essential chaperonin GroES/GroEL are important for the folding of intracellular proteins (118, 119). It should be noted that DnaK, its partners, and Tig are nonessential in B. subtilis; however, the corresponding genes are highly expressed, and they are conserved among the genome-reduced mollicutes. Of note, the nonessential DnaK/DnaJ chaperone is also present in M. mycoides JCVI-syn3.0, whereas the universally conserved essential GroES/GroEL chaperonin is not (10). In addition, viability of B. subtilis requires the presence of one intramembrane quality control protease, and we selected HtrB for this activity (120, 121).

Central Carbon Metabolism

Central carbon metabolism is at the heart of any cellular metabolism. We have selected 26 proteins (among them 4 essential proteins) for this function. Glycolysis provides the cell with precursors for further metabolic pathways, ATP for substrate-level phosphorylation, and reducing power to drive respiration (see Fig. 1 and 3 for details). Moreover, the pentose phosphate pathway generates reducing power for anabolic reactions, erythrose-4-phosphate as a precursor for chorismate synthesis (Fig. 3), and ribose-5-phosphate for the synthesis of nucleic acids. To generate acetyl coenzyme A (acetyl-CoA) for lipid biosynthesis, pyruvate dehydrogenase is required (122, 123). Moreover, the acetyl-CoA synthetase recycles the acetate derived from cell wall biosynthesis (124) (Fig. 3 and 10) (see below). Both glycolysis and the pentose phosphate pathway generate a large amount of reducing power. NAD⁺ can be regenerated in respiration, and the YtsJ/MalS transhydrogenation cycle is used for balancing NADPH⁺ levels (Fig. 3) (125). The minimal cell does not need the citric acid cycle. This pathway can be completely deleted in B. subtilis (our unpublished results). The minimal organism M. mycoides JCVI-syn3.0 encodes the enzymes for glycolysis but only a strongly reduced set (three out of seven) enzymes of the pentose phosphate pathway. Moreover, these cells lack the citric acid cycle. The absence of a full pentose phosphate pathway and of the citric acid cycle is characteristic of the reduced metabolism of Mycoplasma species and reflects their reliance on the uptake of nutrients from the environment (117). Interestingly, of the glycolytic enzymes, the classical fructose 1,6-bisphosphate aldolase is missing in M. mycoides JCVI-syn3.0. The cleavage of fructose 1,6-bisphosphate may be catalyzed by the paralogous IolJ aldolase in these bacteria. It is worth noting that the nearly complete match of the gene sets for glycolysis in the suggested MiniBacillus genome and in M. mycoides JCVI-syn3.0 is exceptional among all metabolic pathways.

Respiration/Energy

ATP production is achieved efficiently by the generation of a proton motive force in respiration and its subsequent use to drive ATP synthesis. A total of 16 proteins are involved in these processes, among them 2 essential proteins (Fig. 3). NADH₂ from glycolysis is reoxidized by the respiration chain consisting of NADH dehydrogenase, menaquinone, and the terminal heme-copper cytochrome aa₃ oxidase. It is important to note that B. subtilis is unable to live under aerobic conditions in the absence of both terminal quinol oxidases (126). We have selected this respiration chain because it is a minimal chain and because the selected terminal oxidase is capable to pumping protons to energize the membrane. Moreover, loss of the heme-copper cytochrome aa₃ oxidase results in impaired growth (126). Next, the multisubunit ATPase uses the proton gradient to provide the cell with ATP. Interestingly, several subunits of the ATPase in M. mycoides JCVI-syn3.0 seem to be too poorly conserved with those of B. subtilis to allow detection by sequence comparison.

Amino Acids

Acquisition of amino acids is one of the major activities of any living cell. This can be achieved by either the uptake of external amino acids, the uptake and subsequent degradation of peptides, or the biosynthesis of amino acids. As biosynthesis usually involves significantly more proteins than does uptake, we included mainly transport systems for amino acids in the list (see also Fig. 1 and 4 for details). Exceptions are the biosyntheses of alanine, glycine, serine, phenylalanine, tyrosine, asparagine, and glutamine. The single glycine biosynthetic enzyme (serine hydroxymethyltransferase [GlyA]) is essential, suggesting that B. subtilis has to synthesize this amino acid. For alanine, serine, asparagine, and the aromatic amino acids, no transporter is known. For glutamine, biosynthesis requires only one enzyme (glutamine synthetase [GlnA]), and the only known glutamine transporter is composed of several subunits and is expressed only during sporulation. Among the 30 proteins that are required for amino acid acquisition in the frame of the MiniBacillus genome, only 2 (the essential GlyA protein and a cysteine transporter) are also present in M. mycoides JCVI-syn3.0, suggesting that this organism acquires amino acids in a completely different way. Likely, the latter organism relies on the transport and intracellular degradation of oligopeptides to obtain amino acids (10).

Nucleotides/Phosphate

Complex media like LB cannot meet the demand of B. subtilis for nucleotides (R. Switzer, personal communication). Therefore, the minimal cell has to carry 35 genes for nucleotide de novo synthesis. Of these genes, 11 are essential. Both purine and pyrimidine biosyntheses are initiated by the synthesis of phosphoribosylpyrophosphate from ribose-5-phosphate (catalyzed by the essential and universally conserved phosphoribosylpyrophosphate synthetase Prs). The pathways of nucleotide biosynthesis are schematically shown in Fig. 1 (for more details, see Fig. 5). The pyrimidine and purine nucleotide biosynthetic pathways converge with the reduction of ribonucleotides by the NrdEIF complex and the final formation of nucleoside triphosphates by the nucleoside diphosphate kinase Ndk (127). For one of the essential proteins implicated in nucleotide biosynthesis, HprT, the reason for its essentiality is unclear (128).

One protein is required for phosphate acquisition. Pit is a constitutively expressed low-affinity phosphate transporter (Fig. 2).

Lipids

Lipid biosynthesis is an essential function in most bacterial cells. The pathway starts with the carboxylation of acetyl-CoA derived from glycolysis (Fig. 1) and ends with the addition of a so-called head group to phosphatidic acid. Fatty acid biosynthesis is an essential process in nearly all bacteria, but this pathway is lacking in Mycoplasma species (129). Accordingly, fatty acid biosynthetic genes are absent from the minimal genome of M. mycoides JCVI-syn3.0. These organisms rely on fatty acid acquisition from their hosts or the provided complex medium (10). In total, this pathway requires 19 genes (Fig. 6). With the exception of fabHA and fabI, all of these genes are essential in B. subtilis. The two nonessential genes in this pathway have functional paralogs, and it is known that the encoded functions are essential; i.e., one of the paralogs has to be present (36). The pathway is initiated by the synthesis of malonyl-CoA from acetyl-CoA by the four-subunit enzyme complex acetyl-CoA carboxylase. It should be noted that AccB, one of the proteins of this complex, needs to be biotinylated by the essential biotin protein ligase BirA (130). After the formation of acetoacetyl-acyl carrier protein (ACP), the cycle of consecutive fatty acid elongation involves FabG, YwpB, FabI, and FabF. The formation of phospholipids is initiated by the replacement of the acyl carrier protein by a phosphate group and the subsequent addition of glycerol-3-phosphate. After the addition of the second fatty acid, the head group is attached to form phosphatidylglycerol. It should be noted that the enzyme catalyzing the final reaction of the pathway, the dephosphorylation of phosphatidylglycerol phosphate, has not yet been identified in B. subtilis. The lipids of B. subtilis 168 contain up to five different head groups (131). However, it has been established that membranes consisting of only phosphatidylglycerol do not confer any growth disadvantages (131). Therefore, we decided to keep only this head group.

Cofactors

B. subtilis needs 11 distinct cofactors, i.e., NAD, flavin adenine dinucleotide (FAD), pyridoxal phosphate, biotin, thiamine, lipoic acid, coenzyme A, S-adenosylmethionine, folate, heme, and menaquinone. For some of these molecules, their acquisition is possible by uptake and biosynthesis. For the minimal cell, we have chosen to include uptake whenever possible (nicotinate and riboflavin as precursors for NAD and FAD, respectively; biotin; and thiamine) (see Fig. 1 and 7 to 9 for details). As a result, 62 proteins are required for the acquisition of cofactors. Of these proteins, those necessary for the synthesis of NAD from nicotinate, the two subunits of the pyridoxal-5-phosphate synthase, the lipoic acid synthase, the enzyme for the final step of CoA synthesis, the S-adenosylmethionine synthetase, the dihydrofolate reductase, and the enzymes for menaquinone synthesis are essential (a total of 14 proteins). It is worth noting that MenH, an enzyme of the menachinone biosynthetic pathway, was recently shown to be dispensable (132, 133). While none of the cofactor biosynthetic pathways are present in Mycoplasma species, and thus are also lacking in M. mycoides JCVI-syn3.0, this minimal genome possesses all three general components of the so-called ECF (energy-coupling factor) transporters that are capable of transporting several cofactors (10). The substrate-specific S proteins for these transporters are generally poorly conserved and barely detectable in sequence comparisons (134, 135).

Metals and Iron-Sulfur Clusters

Many enzymes and proteins need metal ions for activity. Moreover, potassium and sodium are important for the osmotic stability of the cell. The minimal B. subtilis cell has to transport sodium, potassium, iron, magnesium, manganese, zinc, and copper ions. With the exception of iron, we have included one transporter (usually of low affinity) for each ion. For iron uptake, the minimal cell should possess the EfeUO system for elemental iron uptake and the iron-citrate ABC transporter YhfQ-YfmCDEF (136, 137). Sodium is imported by amino acid-sodium symporters. It has to be exported by the Mrp complex (138) (Fig. 2). Surprisingly, in the genome of M. mycoides JCVI-syn3.0, only a potassium transporter and two putative transporters for magnesium have been annotated (10). The identities of other metal transporters in the minimal cell thus remain unclear.

Many proteins involved in redox reactions need iron-sulfur clusters for their activity. The synthesis of these clusters from cysteine and ferrous iron and their attachment to proteins involve seven proteins, five of which are essential and are therefore included (Fig. 7) (139).

Cell Wall Biosynthesis

Cell wall biosynthesis is intimately linked to cell division. First, we briefly discuss the set of enzymes that is involved in catalytic activities for the cell wall components, the peptidoglycan, and the teichoic and lipoteichoic acids. Fifty-five proteins (43 essential proteins) are required for these pathways.

Peptidoglycan synthesis starts with the synthesis of glucosamine-6-phosphate from glutamine and fructose-6-phosphate (140). The next important intermediate is UDP-N-acetylglucosamine (UDP-GlcNAc). This compound is converted to UDP-N-acetylmuramic acid, to which the 5 amino acids of the peptide are subsequently attached. The peptide contains three unusual amino acids, d-glutamate, meso-diaminopimelic acid, and d-alanine, which have to be provided by the action of racemases (d-Glu and d-Ala) or by a biosynthetic pathway starting from aspartate. The enzyme MraY replaces the activating UDP moiety by undecaprenyl phosphate, which has to be synthesized starting from pyruvate and glyceraldehyde-3-phosphate (see Fig. 1 and 10 for details). N-Acetylglucosamine is added to give rise to a molecule called lipid II (141). Owing to the undecaprenyl phosphate moiety, this molecule can integrate into the membrane and is then flipped to the outer side by the action of a flippase, MurJ. Most of the enzymes participating in the pathways mentioned above are essential. MurJ is an exception, as B. subtilis also possesses the weakly expressed functional paralog Amj (142).

Lipid II is the functional unit of peptidoglycan polymerization driven by the penicillin-binding proteins (141). These proteins catalyze the consecutive elongation of the peptidoglycan chain as well as the introduction of cross-links between the peptides. Moreover, autolysins are necessary to introduce breaks in the molecule that serve as targets for the introduction of new material. Penicillin-binding proteins and autolysins are present with several paralogs in B. subtilis. For the minimal cell, we have selected penicillin-binding proteins 1 (PonA), 2B (PbpB), and 2A (PbpA) and the autolysins LytE and LytF (143 –147). As outlined above, this selection was made according to their expression profiles and the dependence on other proteins. As an example, there is a functional paralog of LytE, CwlO (148). For the activity of CwlO, B. subtilis also needs the ABC transporter FtsEX and the small protein Mbl (149). Thus, the choice of LytE allowed a smaller number of genes.

Another essential component of the Gram-positive cell wall is teichoic acids. In B. subtilis, these molecules can be attached to either peptidoglycan or the membrane via a lipid anchor. Wall teichoic acid is polyglycerol phosphate attached to the peptidoglycan via a disaccharide anchor (150). The pathway is initiated by the synthesis of the anchor from undecaprenyl phosphate and activated N-acetylglucosamine and N-acetylmannosamine (UDP-GlcNAc and UDP-N-acetylmannosamine [UDP-ManNAc]). Glycerol is activated by the synthesis of CDP-glycerol. This compound serves as the substrate for the initial addition of glycerol to the undecaprenyl phosphate-carrying disaccharide and then for consecutive rounds of chain elongation. The polymer is then exported by the TagGH ABC transporter and is finally attached to peptidoglycan by TagU (Fig. 11) (150). Lipoteichoic acid (LTA) is polyglycerol phosphate attached to the membrane via a β-diglucosyl-diacylglycerol anchor. This anchor is synthesized from glucose-6-phosphate and diacylglycerol by the enzyme UgtP (151). This anchor has to be flipped to the outside of the membrane by an as-yet-unknown flippase. The lipoteichoic acid synthase LtaS then processively adds glycerol phosphate moieties using phosphatidylglycerol, the building block of the lipid bilayer, as the substrate. This reaction releases toxic diacylglycerol, which is recycled by the essential diacylglycerol kinase DgkB to phosphatidic acid, which is used for the synthesis of phosphatidylglycerol (see above) (Fig. 6) (32, 152, 153).

Cell Division

Twenty-two proteins are necessary for the functions of cell division and cell shape determination. Of these proteins, nine are essential. Most of these proteins are part of several large protein complexes, i.e., the divisome, which links cell division to cell wall synthesis, and the elongasome, which recruits the cell wall biosynthetic enzyme to the lateral cell wall. Accordingly, cell wall biosynthetic enzymes (see above) are part of these complexes. This is the case for MraY, MurF, MurG, and penicillin-binding proteins 1 and 2B (PonA and PbpB, respectively), which are part of the divisome and/or the elongasome (154, 155). To achieve the correct placement of the division site, DivIVA in concert with the proteins of the Min system prevent the formation of the Z ring close to nascent division sites and cell poles (156, 157). Finally, WalJ coordinates cell division and DNA replication (158). Interestingly, the essential tubulin-like cell division initiation protein FtsZ has long been regarded as being present in all organisms. However, this protein is absent in some Mycoplasma species and is nonessential in those Mycoplasma species that encode it (159). Accordingly, FtsZ is also not encoded in the genome of M. mycoides JCVI-syn3.0 (10). Moreover, FtsZ is dispensable in B. subtilis L-form cells that are capable of growing without a cell wall (160, 161). However, as these cells grow very slowly and are fragile, we deemed the FtsZ-dependent cell division pathway essential in the context of the MiniBacillus genome.

Signaling in Cell Division

As in other bacteria, most signal transduction systems of B. subtilis are dispensable. This is the case for all alternative sigma factors as well as for classical transcription repressors and activators. However, there are two notable exceptions, and they are both involved in cell wall metabolism and cell division. The WalRK two-component regulatory system in B. subtilis and related Gram-positive bacteria is the only two-component system known to be essential. This system allows the expression of genes for cell division and the synthesis of wall teichoic acid, such as lytE, mreBH, ftsZ, and the tag genes (162, 163). Moreover, cyclic di-AMP (c-di-AMP) is a unique essential second messenger. The precise reason for the essentiality of this nucleotide is unknown, but c-di-AMP has been implicated in cell division and cell wall biosynthesis (164, 165). Interestingly, c-di-AMP is not only essential but also toxic if it accumulates (166). Therefore, CdaA was selected among the three diadenylate cyclases in B. subtilis, and GdpP was selected as one of two phosphodiesterases that degrade the second messenger c-di-AMP.

Integrity of the Cell (Protection and Genome Integrity)

All the functions discussed above are essential for the minimal cell. However, a minimal organism also needs activities that are not per se essential but that help to keep and protect the integrity of the cell. This involves protection against toxic metabolites and intermediates as well as against damage caused by oxygen (Fig. 2). In addition, the genome of the minimal cell has to be sufficiently stable. For this purpose, proteins involved in DNA repair and genome integrity are important components of any minimal cell that is intended for stable reproduction. In the list of required proteins, we have included eight proteins for protection and genome integrity.

Additional Proteins of the Minimal Cell

There are 11 additional proteins that may be important for a minimal cell. Two of these proteins (PpaC and YlaN) are essential, but their precise function is not known. Moreover, based on our own experimental data and those of colleagues, YitI, YitW, and YqhY are important for viability (P. Dos Santos, personal communication; our unpublished results). Four proteins (YkwC, YlbN, YpfD, and YugI) are very strongly expressed (39) and therefore certainly of vital importance. FloT is involved in the control of membrane fluidity, and the loss of both flotillin-like proteins is known to result in severe defects in cell morphology and transformation (167). Finally, the nonessential GTPase YyaF is highly conserved in Gram-positive bacteria, even among all groups of mollicutes (42). Due to its GTPase activity, it is tempting to speculate that YyaF plays a role in ribosome assembly or in translation.

Open Questions

Even though B. subtilis is one of the best-studied and best-understood organisms, there are still gaps in our knowledge. These gaps pose serious challenges to any minimal genome project. Several of these uncertainties are mentioned above. In particular, we have identified gaps in the pathways of phospholipid biosynthesis (the final phosphatase) and lipoteichoic acid synthesis (the flippase for the membrane anchor). Concerning phosphatase, there are several uncharacterized phosphatases encoded in the B. subtilis genome, leaving room for further research.

A general uncertainty is the disposal of toxic or harmful compounds. Even though we have included eight proteins for this activity, this list may be far from being complete. Another function with significant gaps in our knowledge is RNA modification. While our list is based on current knowledge, it is possible that not all of these modifications are truly essential. Moreover, some modifications might become important if other modifications are lacking due to the absence of the responsible enzymes.

Finally, only little is known about essential noncoding RNAs and essential structural DNA regions in B. subtilis. The importance of these features has been uncovered in a recent saturating-mutagenesis study with M. pneumoniae (8).

A MODEL OF MiniBacillus METABOLISM

The selection of functions, genes, and pathways presented above is like a bet. To ensure that this set allows the cell to function, we have developed a model of its metabolism. This model is schematically depicted in Fig. 1, and details are provided in Fig. 2 to 11. With this compilation of metabolic pathways, it becomes obvious that all metabolites that are needed as precursors are produced from simple building blocks. These molecules in turn can be obtained from the medium. On the other hand, all compounds that are produced in the pathways and that are not needed in other reactions are disposed of by specific reactions and pathways.

MiniBacillus AS A STARTING POINT TO DISCOVER AND STUDY NOVEL ANTIMICROBIAL DRUG TARGETS

B. subtilis is closely related to many important pathogens, including Staphylococcus aureus, Listeria monocytogenes, and Streptococcus pyogenes. The genetic complement of MiniBacillus likely represents a set of highly important genes for all of these organisms, suggesting that this set is an excellent starting point to identify promising novel potential antimicrobial drug targets. Indeed, the conserved essential diadenylate cyclase was recently proposed to be a potential drug target, screening systems have been set up, and a first drug has been discovered (168 –170).

For drug targets, knowledge of the protein structure is highly beneficial to facilitate in silico screening of virtual compound libraries. Of all 523 proteins that are encoded by our proposed minimal genome, 471 (90.1%) have a known structure. This figure illustrates the huge progress made by structural biologists, especially in the framework of large structural genomic projects (171). The unique combination of high structural coverage and excellent prior knowledge of the protein components of MiniBacillus makes the list of proteins presented in Table 2 a good starting point to identify novel potential targets. Moreover, the remaining 10% of the proteins are an important challenge for structural biologists.

Knowing all the structures of the proteins and even protein complexes of MiniBacillus not only is important in the context of the search for novel drugs but also is by itself a major scientific goal. With knowledge of the structure of all the components of a minimal cell, we can start making a real image of what is going on in the cell and thus become much closer to the answer of the old question, “What is life?”

EXPERIMENTAL APPROACH TO THE CONSTRUCTION OF MiniBacillus

To obtain a minimal cell, MiniBacillus, with the described set of genes, the top-down approach seems to be the most appropriate. Such an approach requires efficient systems for markerless gene deletions, which in turn indicates a need for a highly efficient genetic system. B. subtilis is known for its natural competence. This system is under the control of a central transcription factor, ComK. In order to be able to perform consecutive deletions, the genes involved in genetic competence have to be kept until the very end of the successive genome reduction process. Therefore, another set of 54 genes has to be considered (see Table S1 in the supplemental material). Optimally, expression of competence genes would occur not just in 10% of the population as in the B. subtilis wild-type strain but in all cells of a population. For this purpose, a supercompetent B. subtilis strain was recently engineered (172). In this strain, the transcription factor ComK and ComS, a protein which prevents the proteolytic degradation of ComK, are expressed under the control of a strong mannitol-inducible promoter.

Several systems have been proposed for the construction of markerless deletions. The Cre-Lox system is very efficient, and it has been adapted to B. subtilis (173) but has the disadvantage of leaving scars behind. Such scars would accumulate if dozens of deletions were performed. Alternative systems rely on the integration of genes, which become toxic under defined conditions with a deletion cassette. Counterselection then allows the rapid and specific loss of the cassette, i.e., the markerless deletion of the desired regions. Several counterselection systems have been implemented for B. subtilis. The most popular systems rely on the mazF RNase; the upp gene, which is counterselected in the presence of 5-fluorouracil; and the mannose transporter ManP, which generates toxic mannose-6-phosphate (19). Using the latter system, a significant reduction of the B. subtilis genome was recently achieved (19, 174, 175).

In B. subtilis, many important genes that are part of the minimal gene set listed in Table 2 are scattered on the chromosome. Therefore, defragmentation of such regions will facilitate the effective progress of a genome reduction program. For this defragmentation, desired fragments can be assembled and introduced into the chromosome by using, e.g., Gibson assembly or the ligase cycling reaction in combination with the markerless deletion methods mentioned above (176, 177).

FINAL REMARKS

There is not a single solution for the question of a minimal genome, but there are many possible answers. To fully understand the functionality of living cells, minimal organisms based on different model organisms are required. Thus, the construction of M. mycoides JCVI-syn3.0 marks an important breakthrough, but with its large number of unknown genes, it cannot provide the full picture (10). It will be essential to continue genome reduction projects with very different bacteria, yeasts, and even archaea to be able to finally draw a conclusion about the essence of life.

(For more information, see http://www.minibacillus.org/.)

Supplementary Material

Supplemental material

supp_80_4_955__index.html^{(808B, html)}

ACKNOWLEDGMENTS

We are grateful to many friends and colleagues from the Bacillus scientific community who provided information on minimal gene sets required for specific functions. We give our special thanks to Valerie de Crécy-Lagard, Patricia dos Santos, Juan Alonso, Richard Daniel, Jan Maarten van Dijl, David Dubnau, Stephan Gruber, Sven Halbedel, Colin Harwood, John Helmann, Laurent Janniere, Oscar Kuipers, Mohamed Marahiel, Lyle Simmons, and Robert Switzer. We acknowledge Sarah Wilcken and Raphael Michna for the help with bioinformatic issues.

D.R.R. was supported by the Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences (GGNB) (DFG grant GCS226/2). J.S. was supported by the Deutsche Forschungsgemeinschaft through grant SPP1879.

Biographies

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Daniel R. Reuß studied microbiology and biochemistry at the Georg August University Göttingen, Germany. During his studies, he did an internship at DSM Nutritional Products in Basel, Switzerland, where he worked on vitamin B₆ production in Bacillus subtilis. After his graduation in 2012, he worked with Prof. Ken-ichi Yoshida at Kobe University, Japan, and focused on work with the inositol pathway in B. subtilis. Afterwards, he returned to Göttingen, where he is now doing his Ph.D. working on the B. subtilis genome reduction project.

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Fabian M. Commichau studied biology at the RWTH, Aachen, Germany, and obtained a Ph.D. in microbiology at the University of Göttingen, Germany. After postdoctoral work on protein-protein interactions at the University of Göttingen and at the University of Basel, Switzerland, he stayed for 2 years in industry, R&D, at DSM Nutritional Products Ltd., Kaiseraugst, Switzerland. In 2011, he returned to the University of Göttingen, where he became a group leader at the Department of General Microbiology at the Institute for Microbiology and Genetics and obtained his habilitation. In 2016, his interests are amino acid and vitamin metabolism in Gram-positive bacteria, bacterial evolution and molecular mechanisms underlying genome instability, second-messenger signaling in the human-pathogenic bacterium Listeria monocytogenes, and minimal genomes.

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Jan Gundlach studied biology at the University of Göttingen, Germany. During his studies, he was working on protein-protein interactions and second-messenger signaling in Bacillus subtilis. He was also part of a group of students that participated in the iGEM competition in 2013, which was organized and held at MIT, Boston, MA. Since 2014, he has been a Ph.D. candidate at the University of Göttingen, Germany. In 2016, his interests are second-messenger signaling and ion homeostasis in Gram-positive bacteria.

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Bingyao Zhu finished her bachelor's study in biology at China Agricultural University, China. Later, she pursued a master's degree in microbiology and biochemistry at the University of Göttingen, Germany. She was member of a group of students that participated in the iGEM competition in 2013, which was organized and held at MIT, Boston, MA. After obtaining her master's degree, she became a Ph.D. candidate at the Department of General Microbiology, University of Göttingen. She is currently working on the further development of SubtiWiki, an integrated annotation database for Bacillus subtilis, as her Ph.D. project. She is interested in developing new tools to collect and organize data, in order to find inner connections.

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Jörg Stülke studied biology at Ernst Moritz Arndt University, Greifswald, Germany, and obtained a Ph.D. in microbiology at the University of Greifswald. After postdoctoral work on carbon catabolite repression in Bacillus subtilis at the University of Lund, Sweden, and the Institut Pasteur, Paris, France, he became a group leader at the Chair of Microbiology at the University of Erlangen, Germany. He obtained his habilitation on the control of carbon metabolism in Bacillus subtilis in 2000. In 2003, he was appointed Full Professor for Microbiology at the Georg August University Göttingen, Germany. He studies RNA-mediated control of gene expression by RNA switches and RNA degradation. Moreover, he is interested in heterogeneous gene expression and in the discovery of the function of the essential second messenger cyclic di-AMP. In the framework of the minimal genome project, he is developing tools for genome annotation and involved in the construction and analysis of genome-reduced strains of B. subtilis.

Footnotes

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MMBR.00029-16.

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PERMALINK

The Blueprint of a Minimal Cell: MiniBacillus

Daniel R Reuß

Fabian M Commichau

Jan Gundlach

Bingyao Zhu

Jörg Stülke

SUMMARY

INTRODUCTION

CONSIDERATIONS FOR THE DEFINITION OF THE GENE SET FOR A MINIMAL CELL

THE GENETIC COMPLEMENT OF A MINIMAL CELL

TABLE 1.

FIG 1.

FIG 2.

FIG 3.

FIG 4.

FIG 5.

FIG 6.

FIG 7.

FIG 8.

FIG 9.

FIG 10.

FIG 11.

TABLE 2.

TABLE 3.

DNA Replication and Chromosome Segregation/Maintenance

Transcription

mRNA Folding and Degradation

Translation

Protein Secretion

Intracellular Chaperones, Protein Quality Control, and Proteolysis

Central Carbon Metabolism

Respiration/Energy

Amino Acids

Nucleotides/Phosphate

Lipids

Cofactors

Metals and Iron-Sulfur Clusters

Cell Wall Biosynthesis

Cell Division

Signaling in Cell Division

Integrity of the Cell (Protection and Genome Integrity)

Additional Proteins of the Minimal Cell

Open Questions

A MODEL OF MiniBacillus METABOLISM

MiniBacillus AS A STARTING POINT TO DISCOVER AND STUDY NOVEL ANTIMICROBIAL DRUG TARGETS

EXPERIMENTAL APPROACH TO THE CONSTRUCTION OF MiniBacillus

FINAL REMARKS

Supplementary Material

ACKNOWLEDGMENTS

Biographies

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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