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identify genes that are essential for cell viability. The era of
genome sequencing and comparative genomics produced a
series of estimates of the composition of a minimal cell and
how it would function [3–7]. A plethora of model organisms
have been investigated, including a broad spectrum of
obligatory host-associated bacteria that possess near-min-
imal genomes as a result of evolutionary gene erosion.
These analyses led to the identification of quite similar,
albeit non-identical, sets of genes [3–18] (Table 1). As a
consequence, there is presently no consensus about which
genes are really essential. We believe that it is safe to
assume that some genes are absolutely necessary for the
survival of any life form, whereas others can be omitted (at
least in the laboratory environment). We focus here on the
consists of less than 40 genes [7]. This number of genes
identified by comparative genomics is artificially small,
mainly because many essential cellular functions can be
encoded by non-orthologous genes. Given that gene se-
quence homologies are lower between phylogenetically
unrelated organisms, the evolutionary distance between
analyzed genomes can have a significant impact on the
outcome of comparative genome analyses. In the first
experimental study, saturation transposon mutagenesis
(Figure 1b) was used in M. genitalium to identify approxi-
mately 300 genes that could not be inactivated by trans-
poson and thus were considered to be essential [21]. Since
then this approach has been used in a broad spectrum of
organisms and has led to the identification of essential
gene sets of varying sizes (Table 1). This variation is
probably because saturation transposon mutagenesisE-mail addresses: Juhas, M. (mario.juhas@uzh.ch, mariojuhas@googlemail.com).
Essence of life: esse
minimal genomes
Mario Juhas1, Leo Eberl1 and John I. Gla
1Department of Microbiology, Institute of Plant Biology, Unive
2Synthetic Biology and Bioenergy group, The J. Craig Venter In
Essential genes are absolutely required for cell survival.
Determination of the universal minimal set of genes
needed to sustain life is, therefore, expected to contrib-
ute greatly to our understanding of life at its simplest
level, with applications in medicine and synthetic biolo-
gy. The search for the minimal genome has led to the
identification of often variable gene sets. We argue here
that, based on the outcome of these analyses, it is
becoming increasingly evident that some genes, and
the functions encoded by them, are absolutely necessary
for the survival of any living entity, whereas others can
be omitted. We also examine ways of determining the
minimal genome and discuss possible practical applica-
tions of a minimal cell.
Essential genes
Essential genes are, by definition, absolutely required for
the survival of an organism and are therefore considered
the foundation of life. Identification of the universal mini-
mal set of essential genes has been the pursuit of biologists
for two main purposes. With the advent of the first micro-
bial genome sequences, the pharmaceutical industry hy-
pothesized that genes encoding essential cellular functions
would be logical targets for new antibiotics, due to their
indispensability for bacterial cell survival [1]. Another
group of scientists, led initially by Max Delbru¨ ck, saw
the identification of essential genes as a route that could
lead to an understanding of the universal principles of life
[2]. The latest to take up the question of essential genes are
researchers in the emerging science of synthetic biology,
who aim to create a cell with a minimal genome.
A wide variety of approaches have been employed to
562 0962-8924/$ – see front matter � 2011 Elsevier Ltd. All rights rese
ntial genes of
2
 of Zurich, CH-8057 Zurich, Switzerland
tute, Rockville, MD 20850, USA
biological aspects of minimal genomes and discuss func-
tions encoded by essential genes that are required to
support cellular life. We examine methods exploited in
the search for the universal minimal genome and investi-
gate reasons why they have led to such variation in the sets
of genes identified. We also discuss possible practical
applications of a minimal cell – in medicine for the identi-
fication of novel antimicrobial targets and in synthetic
biology for the creation of specialized cells with desired
phenotypic traits.
Experimental and computational methods to assess the
minimal genome
Several approaches have been employed to identify genes
that are essential for cell viability. As the amount of data
increased it became clear that there was variation in the
sets of essential protein-coding genes identified (Table 1).
One of the challenges currently is to determine which of
these identified genes are really essential.
Why is there such variation in the identified sets of
essential genes? First, none of the methods used for their
identification is flawless. An initial attempt to identify a
minimal genome was performed using comparative geno-
mics, assuming that genes shared between organisms are
likely to be essential (Figure 1a). This analysis was stimu-
lated by the availability of the first sequenced genomes of
Haemophilus influenzae [19] and Mycoplasma genitalium
[20] and led to the identification of approximately 250
candidate essential genes [3]. As more genome sequences
became available, the number of shared genes identified by
comparative genomics decreased (Table 1). As a conse-
quence, it was concluded that the universal conserved core
rved. doi:10.1016/j.tcb.2011.07.005 Trends in Cell Biology, October 2011, Vol. 21, No. 10
ssen
counts as essential those genes that only slow down growth
without arresting it. Furthermore, transposon bombard-
ment is not entirely random – an analysis using two
variants of the same transposon with different antibiotic
resistance genes can yield slightly different essential gene
sets (J. G. et al., unpublished). Recently, saturation trans-
poson mutagenesis coupled with next-generation sequenc-
ing has been used to facilitate high-throughput
identification of essential genes (Figure 1c) [13]. Other
frequently used methods include the systematic inactiva-
tion of individual genes or the use of antisense RNA
(Figure 1d,e) [14–18]. The use of antisense RNA is limited
to those genes for which an adequate expression of the
inhibitory RNA can be obtained. In addition, inactivating
individual genes means that essential genes that are
present in more than one copy will be missed. Experimen-
tal genome reduction, based on random or targeted remov-
Table 1. Essential genes identified by different methods
Number of protein-coding e
Organism
Saccharomyces cerevisiae 878 
Haemophilus influenzae 642 
Acinetobacter baylyi 499 
Mycoplasma pulmonis 461 
Mycoplasma genitalium 381 
Salmonella enterica Typhi 356 
Pseudomonas aeruginosa 335 
Helicobacter pylori 323 
Escherichia coli 303 
Staphylococcus aureus 302 
Bacillus subtilis 271 
Numbers of genomes compared
2 256 
5 180 
7 156 
100 63 
147 35 
Opinion
al of genomic segments, represents another avenue for
minimal genome identification (Figure 1f). This method
has led to the successful elimination of approximately 10–
30% of the Escherichia coli genome without any detectable
defect in bacterial viability [22–24]. The main limitation of
random genome reduction is the need for subsequent
resequencing of the genome to identify the exact location
of the deletion, and targeted genome reductions are cur-
rently still unable to generate a minimal genome.
In addition to variation due to methodological differ-
ences, the organism investigated also impacts upon the
outcome of the analysis. For instance, the set of essential
genes of H. influenzae consists of almost twice as many
genes as that of Helicobacterpylori identified by the same
method (Table 1). It is therefore very likely that these
variations reflect the varying environmental conditions
and lifestyles of the organisms. In other words, in addition
to a universal minimal genome, other important genes
could also be essential for the survival of a particular
organism or in a specific environment.
One of the main challenges for future research is to
resolve the variation in the sets of essential genes identi-
fied depending on which method of analysis is employed.
To address this issue, essential genes in an organism could
be first identified by comparative genomics and their es-
sentiality confirmed by experimental methods. Alterna-
tively, genes found to be essential in one organism could
be investigated in other species, preferably by different
experimental methods. Currently used methods should be
modified to minimize their weaknesses. For example, syn-
thetic lethality screens, which use mutants to search for
other mutations further attenuating viability, could be
used to identify essential genes present in more than
one copy [25,26]. More in-depth studies are required to
determine those genes that are essential for the survival of
any living organism. In the quest to find the universal core
set of essential genes, researchers have also turned to
organisms in which nature has reduced their genomes
close to the minimum.
tial genes Method of identification Ref.
Single-gene-specific mutagenesis [14]
Saturation transposon mutagenesis [8]
Single-gene-specific mutagenesis [15]
Saturation transposon mutagenesis [12]
Saturation transposon mutagenesis [10]
Saturation transposon mutagenesis [13]
Saturation transposon mutagenesis [11]
Saturation transposon mutagenesis [9]
Single-gene-specific mutagenesis [16]
Antisense RNA inhibition [17]
Single-gene-specific mutagenesis [18]
Comparative genomics [3]
Comparative genomics [4]
Comparative genomics [5]
Comparative genomics [6]
Comparative genomics [7]
Trends in Cell Biology October 2011, Vol. 21, No. 10
Essential genes in naturally occurring minimal genomes
A broad spectrum of near-minimal genomes can be found in
nature as a result of reductive evolution, which eliminates
genes non-essential for survival in a particular environ-
ment. The smallest genome identified to date is that of
Hodgkinia cicadicola, a symbiont of cicadas, which is only
144 Kb in size and potentially contains 188 genes [27]. The
extremely small genomes of endosymbionts usually encode
only the most basic processes, suggesting that some of their
genes might have been transferred into the host cell nu-
cleus. The endosymbiont Wolbachia, for instance, transfers
1 Mb fragments to the nucleus of its host [28]. The genome
of H. cicadicola harbors even fewer genes than some chlor-
oplasts; however, comparative analyses have shown that
the gene sets of endosymbionts and those of cellular orga-
nelles differ significantly, because endosymbionts still har-
bor genes involved in replication, transcription and
translation [29].
How can endosymbionts survive despite their extremely
small genomes? Phylogenetic studies show that endosym-
bionts and their free-living relatives evolved from the
same free-living ancestors via genome reduction [29–31].
Genome erosion is possible in endosymbionts due to the
563
Genome 1
Genome 2
Essential
Key:
T
T T T
In silico comparison of genomes
mRNA
Antisense RNA 
Monitoring growth
Non-essential
Essential
Essential
(a)
(b)
(c)
(d)
(e)
(f)
Suicide plasmid 
Monitoring growth
Non-essential
Essential
Disruption of
target gene
G - Gene
C - Colony
T - Transposon
H - Homologous sequence
Cell
Cell
Cell
Chromosome
H1 
H2 
H1 
H1 H2 H2 
Suicide plasmid integration
and homologous recombination Segment
to be deleted 
Chromosome
Monitoring growth
Non-essential segment
Essential segment 
Inhibition of
expression
Growth and sequencing of colonies
T
T T T
Cell
Illumina
Insertion index
Fr
eq
ue
nc
y
1 2 3 4 5 60
Essential
genes
Cultivation and sequencing of cells
with next-generation sequencer
Transposon mutant
library 
Transposon
bombardment
Transposon mutant
library
Transposon
bombardment
G G G G G
G G
G
G
GG
G G G G G G
G
G
G
G
G G G G
G G G G G G C
C C C C
C C C C
C C C C
C CC
G
TRENDS in Cell Biology 
Figure 1. Identification of essential genes. Various methods are used for the identification of essential genes. (a) Comparative genomics compares genomes in silico to
identify essential genes. Genes shared among genomes are considered essential. (b) Saturation transposon mutagenesis exploits transposons for generation of whole-
genome mutant libraries. Mutants are grown on selective media and sequenced to identify disrupted genes. Genes whose disruptions produce viable colonies are non-
essential and the remaining genes are considered essential. (c) Saturation transposon mutagenesis in combination with next-generation sequencing has been used for
high-throughput identification of essential genes. Transposon libraries are sequenced with the help of a next-generation sequencer (e.g. Illumina) and the insertion index
for each gene is calculated by dividing the number of unique insertion sites for any given gene by the gene length. Genes with an insertion index close to 0 are considered to
be essential. (d) Antisense RNA is used to inhibit the expression of a target gene by binding to its mRNA. If the target gene is essential, the cell will not survive. (e) Single-
gene-specific mutagenesis disrupts individual genes by integration of a suicide plasmid. Lethality indicates that a disrupted gene is essential. (f) Experimental genome
reduction is used to delete large segments from the chromosome by homologous recombination between flanking DNA regions and homologous sequences introduced
into the cell, usually by a suicide plasmid. The deleted DNA segment is considered to be non-essential if cells without it survive.
Opinion Trends in Cell Biology October 2011, Vol. 21, No. 10
564
extremely stable nutrient-rich environment provided by
the host, which makes almost all the metabolic genes and
genes involved in adaptation to changing environments
obsolete. The bacterial endosymbionts of insects, which
harbor the smallest genomes sequenced to date, are utterly
dependent on the stable nutrient-rich environment provid-
ed by their hosts [29].
Mycoplasmas, a group of unusual bacteria that lack a
cell wall, have the smallest genomes of organisms capable
of host-independent growth in laboratory media. Although
Mycoplasmas possess very small genomes, they are not
simple organisms. Detailed examination of the proteome,
transcriptome and metabolome [32–34] of Mycoplasma
pneumoniae cells revealed unexpected complexity, similar
to organisms with much bigger genomes such as E. coli. For
example, protein interaction analysis showed that a large
number of M. pneumoniae proteins were part of more than
one cellular machine or protein complex. In spite of such
unexpected complexity, Mycoplasmas remain among the
most important models to study essential genes, mainly
due to their small genomes and easy cultivation. A team at
the J. Craig Venter Institute is currently removing all
genes considered to be non-essential from Mycoplasma
mycoides to build a synthetic version of this organism
[35]. Although M. mycoides has 828 ORFs, its genome
could be potentially reduced to less than 400 genes based
on saturation transposon mutagenesis in M. genitalium
[10]. The resulting synthetic cell will be a platform to
investigate the basic functions and mechanisms of cellular
life. For instance, such a synthetic cell could provide clear-
er understandingof the function of a particular essential
gene or key biological process, unbiased by non-essential
genes. It is important to keep in mind, however, that the
minimal synthetic cell constructed from the M. mycoides
genome will probably also harbor some essential genes
that are specific to M. mycoides. Hence, whereas the crea-
tion of a synthetic cell based on information from one
organism is in process, it will be a more complicated task
to identify the universal minimal cell. One way to approach
the problem is to compile all information on essential genes
and critically assess which functions are likely to be uni-
versally essential.
Cellular functions encoded by essential genes
Although the general consensus view held by the majority
of researchers is that approximately 300 genes are re-
quired to support cellular life, the exact composition of
the universal minimal genome remains unknown. This
number of candidate essential genes is only a rough esti-
mate based on the results of previous analyses (Table 1).
There is no doubt, however, that some genes are absolutely
necessary for the survival of any lifeform. Analyses of the
functional distribution of essential and non-essential genes
show that the category of information storage and proces-
sing, which involves replication, transcription and trans-
lation, is strongly over-represented among the essential
genes [36]. This is also true for the extremely small gen-
omes of obligate endosymbionts which, although differing
to a great extent among themselves, share a set of core
Opinion
genes [27–29,37,38]. These core genes, which are present in
all analyzed organisms, even in the tiniest genomes of
endosymbionts, include those involved in DNA replication,
transcription and RNA processing, aminoacyl-tRNA for-
mation, protein folding, as well as genes encoding transla-
tion factors and ribosomal subunits [29].
In addition, genes involved in cell membrane biogenesis
are among those identified by minimal genome analyses
[8–18]. The cell membrane separates the protoplasm from
the extracellular environment and allows compartmental-
ization of biological processes, thus its essentiality for a
living cell is unquestionable. Furthermore, the evolution of
membrane structures is also considered to be a key step
towards the emergence of life on early Earth [39]. By
contrast, genes encoding a cell wall or the bacterial outer
membrane are only essential for particular types of cells,
and are therefore not considered to be part of the universal
minimal genome. Genes involved in metabolism are gen-
erally less abundant among the identified essential genes
[40]. In our opinion, metabolic independence is not always
a prerequisite of a ‘viable’ minimal genome because a
plethora of endosymbionts lack metabolism-related genes
entirely. The metabolic genes required for cell survival
depend strongly on the environment because many meta-
bolites can be obtained from the host.
In addition to these core gene categories, analyzed
minimal genomes usually harbor genes with no character-
ized homologs in databases [8–18]. What cellular functions
are encoded by these unknown essential genes? Owing to
their indispensability for cell survival, essential genes
cannot be disrupted by standard mutagenesis, and thus
analyzable mutants of these unknown genes are usually
lacking. This problem can be circumvented by replacement
of the native promoter of the gene with one that can
be easily regulated [41]. In previous analysis of the
385 M. genitalium essential candidates, no function could
be ascribed to 95 genes [10]. Today that number is only 75
(J.G. et al., unpublished). Further investigation of these
uncharacterized genes will provide new insights into the
most fundamental biological processes essential for cell
survival.
Although there is variation in the sets of essential genes
identified, it is becoming clear that some genes are abso-
lutely required for the survival of any living cell. These
‘core’ essential genes, which have been identified consis-
tently in all minimal genome analyses, constitute a good
starting point for generation of the universal minimal
genome. More work will be required, however, especially
to unravel the function of unknown genes because they
might pinpoint additional key gene categories indispens-
able for life. More work also will be needed to characterize
those genes that are essential only within a particular
evolutionary lineage. These ‘accessory’ essential genes
are not conserved because different solutions to the same
biological problem (e.g. cell surface biosynthesis) might
exist in different cell types. Consequently, there might
be no single universal minimal cell because it will always
be biased towards a particular evolutionary trajectory.
Intriguingly, several of these accessory essential genes
appear to encode proteins of unknown function (M.J.
et al., unpublished). Because these genes are conserved
Trends in Cell Biology October 2011, Vol. 21, No. 10
within an evolutionary clade they represent potential tar-
gets for the development of antimicrobials that specifically
565
target a particular group of bacteria. Although work on the
identification of the universal minimal genome is still
ongoing, its potential for the development of novel drugs
and for expanding our basic understanding of biology is
already clear.
The power of simple systems: essential genes in
medicine and synthetic biology
Infectious diseases are a major worldwide cause of mor-
bidity and mortality. Resistance against antibiotics is
rising and newspapers frequently report the emergence
of new ‘superbugs’ [42–44]. In the quest for new antibiotics
the pharmaceutical industry poured a huge effort into
identifying essential genes in important human pathogens
at the outset of the genome sequencing era [1]. Every
essential gene in the model human pathogens Streptococ-
cus pneumoniae and Staphylococcus aureus was consid-
ered a potential target. In assembly-line fashion, high-
throughput assays were developed to find compounds that
block the enzymatic function of essential gene products or
that bind to essential proteins of unknown function. To the
chagrin of the industry, this screening failed to find new
antimicrobials with clinical potential [44]. Although this
failure is largely attributed to a lack of appropriate chemi-
cal diversity in the compound libraries screened, it has also
reinforced a view that it might be more productive to
search for drugs interfering with essential genes encoding
machinery, ssrA and smpB, were shown recently to be
indispensable for survival and might represent excellent
targets for novel antimicrobials [45]. Other recent
additions to the family of novel antimicrobials include
peptidomimetics blocking ostA, an essential gene of
P. aeruginosa whose function is currently under investiga-
tion [46], and boronic esters inhibiting the widely con-
served and essential bacterial methyltransferases CcrM
and MenH [47]. These examples demonstrate that investi-
gation of novel essential genes can be an attractive avenue
for developing novel drugs. More work is needed, however,
to identify other essential genes and to distinguish be-
tween universally essential genes and those indispensable
for the survival of a specific group of bacteria.
While the pharmaceutical industry is searching for
essential genes as targets of novel drugs to kill bacteria,
the emerging field of synthetic biology is looking for essen-
tial genes as a way to build and study a living minimal cell.
Two approaches are being employed by synthetic biolo-
gists: top-down, aiming at further simplification of existing
cells by removal of all non-essential genes; and bottom-up,
attempting to create an artificial cell by synthesizing all of
its essential components [48–50]. The major challenges at
the onset of synthetic biology wereattaining fast, cheap
and reliable synthesis of long DNA fragments and genomes
and their ‘booting-up’ (successful expression) in the cell.
First attempts to synthesize DNA were expensive and
 adva
bori
ligo
e. F
 des
Opinion Trends in Cell Biology October 2011, Vol. 21, No. 10
well-understood functions rather than to screen for novel
essential targets. Despite this, there are several examples
where a deep understanding of novel essential genes
has, in fact, led to new antimicrobial candidates. Two
components of the Helicobacter pylori trans-translational
(a)
(b)
Synthesis of
oligonucleotides
In silico design
of genomes
Assembly of
intermediates
in E. coli 
Figure 2. Creation of synthetic minimal cells with desired phenotypic traits. Recent
‘booting-up’ in a cell, have brought us closer to the creation of a minimal cell har
minimal genomes could be designed in silico before chemical synthesis of DNA o
could be assembled in E.coli, followed by assembly of the genome in S. cerevisia
membrane vesicle. (a) Additional genes, operons or larger DNA fragments encoding
synthetic minimal cell. (b) Alternatively, additional DNA segments could be incorpora
genomes.
566
time-consuming. Work over the last few years has led to
several advances in the synthesis of long DNA sequences
and in 2008 synthesis of the first complete, 583 kb long,
genome of M. genitalium was reported [51–56]. An impor-
tant step towards propagation of synthetic genomes and
Genome transplantation
 to a suitable cell 
Complete genome 
assembly
in S. cerevisiae
Synthesis of genes 
encoding the desired 
phenotype
TRENDS in Cell Biology 
nces in synthetic biology, especially in the areas of genome synthesis and genome
ng only specific phenotypic traits. Modifying a previously described method [35],
nucleotides spanning the entire genome. Subsequently, 10 and 100 kb fragments
inally, the synthetic minimal genome could be transplanted into a suitable cell or
ired phenotypes, such as biofuel or antimicrobial production, could be added to the
ted from the outset by in silico design of specific phenotype-encoding minimal
their ‘booting-up’ was achieved when the genomic DNA
from M. mycoides was transplanted into Mycoplasma
capricolum to produce a viable M. mycoides cell [57]. This
experiment showed that whole genomes can be successful-
ly transferred between species and that they are expressed
in the new host cell, thus changing one species into anoth-
er. The exact mechanism by which the transplanted ge-
nome takes over the host cell remains unsolved, however,
and will require further investigation. Furthermore, the
design and synthesis of the entire M. mycoides genome and
Opinion
its transplantation into a M. capricolum – generating
M. mycoides cells controlled only by the synthetic chromo-
some – was reported recently [35,58]. The major difference
between the two experiments is that in the latter the
complete genome of M. mycoides was designed in silico
and synthesized in the lab before transplantation into a
new cell. This achievement brings us closer to one of the
key goals of synthetic biology: building a minimal cell
harboring, in addition to essential genes, only genes encod-
ing a desired phenotype (Figure 2). The practical applica-
tions of designed minimal cells could be immense, ranging
from the production of biofuels to more effective production
of antimicrobials.
Although two major hurdles in the synthesis of genomes
and their ‘booting-up’ have been largely overcome, other
challenges remain. Whereas the design, synthesis and
transplantation of the M. mycoides genome is a good
starting point, the de novo design of a functional minimal
genome will be a major challenge in the future. Currently,
minimized cells generated by experimental genome reduc-
tions play a leading role in biotech applications because
extensive genome deletions can improve particular cellular
functions such as transformation efficiency or protein
yields [24,50]. We believe that the great potential of a
designed cell harboring only the minimal genome lies in
its simplicity. Minimized E. coli cells still harbor many
non-essential genes, and these could, by as yet unknown
routes, influence the designed and desired functions of the
cell.
Concluding remarks
Work over the past few years aimed at investigation of
essential genes has provided evidence that some genes are
required for the survival of any living organism. These
investigations also raised a number of outstanding ques-
tions for future research (Box 1). Recent high-throughput
methods (e.g. saturation transposon mutagenesis coupled
with next-generation sequencing) or combinations of more
traditional approaches could provide answers to some of
Box 1. Outstanding questions
� Which genes are universally required for the survival of all living
cells and which are essential only for particular cell types
(prokaryotes, eukaryotes or even for different bacterial species)?
� How can we minimize the weaknesses of individual methods used
in a search for the universal minimal gene set?
� How important is the environment of a specific cell type in
determining its minimal gene set?
� What are the roles in cell survival of the uncharacterized genes
identified in minimal genome analyses?
these questions. The search for the universal minimal
genome has identified several uncharacterized genes and
we believe that their investigation could offer invaluable
insights into the basic functions required to sustain cellu-
lar life. The exact composition of the universal minimal
genome is still unknown and the role of the environment in
determining cell type- or condition-specific essential genes
is also unclear. It could turn out that the universal minimal
cell does not exist and that a minimal genome will always
be biased towards a particular organism. We are still far
from a complete understanding of the minimal cell; how-
ever, owing to contributions from researchers around the
world we are starting to get our first glimpse of the essence
of life.
Acknowledgements
The authors thank all researchers who have contributed to our
understanding of essential genes. M.J. and L.E. were supported by the
European Commission 7th Framework Programme (NABATIVI) and
J.I.G. by the Office of Science (BER), U.S. Department of Energy
Cooperative Agreement No. DE-FC02-02ER63453.
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689
	Essence of life: essential genes of minimal genomes
	Essential genes
	Experimental and computational methods to assess the minimal genome
	Essential genes in naturally occurring minimal genomes
	Cellular functions encoded by essential genes
	The power of simple systems: essential genes in medicine and synthetic biology
	Concluding remarks
	Acknowledgements
	References