Microbiology (2014), 160, 2341–2351
Review
DOI 10.1099/mic.0.079376-0
Bacillus subtilis and Escherichia coli essential
genes and minimal cell factories after one decade
of genome engineering
Mario Juhas,1 Daniel R. Reuß,2 Bingyao Zhu2 and Fabian M. Commichau2
Correspondence
Mario Juhas
[email protected]
Fabian M. Commichau
1
Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
2
Department of General Microbiology, Georg-August-University Göttingen, Grisebachstr. 8,
37077 Göttingen, Germany
[email protected]
Received 15 May 2014
Accepted 31 July 2014
Investigation of essential genes, besides contributing to understanding the fundamental principles
of life, has numerous practical applications. Essential genes can be exploited as building blocks of
a tightly controlled cell ‘chassis’. Bacillus subtilis and Escherichia coli K-12 are both wellcharacterized model bacteria used as hosts for a plethora of biotechnological applications.
Determination of the essential genes that constitute the B. subtilis and E. coli minimal genomes is
therefore of the highest importance. Recent advances have led to the modification of the original
B. subtilis and E. coli essential gene sets identified 10 years ago. Furthermore, significant
progress has been made in the area of genome minimization of both model bacteria. This review
provides an update, with particular emphasis on the current essential gene sets and their
comparison with the original gene sets identified 10 years ago. Special attention is focused on the
genome reduction analyses in B. subtilis and E. coli and the construction of minimal cell factories
for industrial applications.
Introduction
Essential genes are absolutely vital for an organism’s
survival. In general, genes can be considered essential if
they cannot be individually knocked out under conditions
where most of the required nutrients are provided in the
growth medium and at a temperature which allows the best
growth of the organism. In the case of two bacteria described
in this review, namely Bacillus subtilis and Escherichia coli,
these conditions were Luria–Bertani (LB) medium supplemented with glucose and cultivation at 37 uC (Baba et al.,
2006; Kobayashi et al., 2003). The identification of essential
genes will help to advance our understanding of the
fundamental principles of life (Commichau et al., 2013;
Jewett & Forster, 2010; Juhas et al., 2011; Moya et al., 2009).
Moreover, bacterium-specific essential genes are considered
to be suitable targets for the development of novel
antimicrobials (Bumann, 2008; Juhas et al., 2012a, b).
Finally, essential genes are important for the emerging field
of synthetic biology as they form the building blocks of future
custom-made minimal genomes of minimal cell factories
(Juhas et al., 2013; Nandagopal & Elowitz, 2011; Seo et al.,
2013). A wide variety of experimental approaches, such as
targeted gene deletions, generation of conditional knockout
mutants, genome-wide RNA interference screens and saturation transposon mutagenesis, were used to identify and study
Three supplementary tables are available with the online version of this
paper.
079376 G 2014 The Authors
essential genes (Baba et al., 2006; Boutros & Ahringer, 2008;
Christen et al., 2011; de Berardinis et al., 2008; French et al.,
2008; Kato & Hashimoto, 2007; Kobayashi et al., 2003;
Langridge et al., 2009; Yu et al., 2008). Furthermore, a
number of computational approaches were employed to
define the core essential gene set needed to sustain life (Juhas
et al., 2011, 2012b; McCutcheon & Moran, 2010; Moya et al.,
2009). However, the complete set of essential genes that is
universally required by all organisms is difficult to determine
experimentally and hard to predict. For instance, survival of
the cell in the absence of a particular enzyme does not always
mean that the enzymic function is not needed, as there might
be other enzymes with the same function present in the cell.
Multiple rRNA operons were shown to be required for
efficient growth of B. subtilis (Yano et al., 2013). Moreover,
some proteins can be considered essential because they
protect the cell against harmful metabolites and proteins, such
as phage-encoded toxins. Hence, these genes can lose their
essentiality if the corresponding harmful gene/pathway is
deleted (Durand et al., 2012).
The rod-shaped Gram-positive and Gram-negative bacteria
B. subtilis and E. coli, respectively, are both well characterized and easily amendable to genetic engineering. Furthermore, the comprehensive community-curated databases
(SubtiWiki, EcoliWiki, BsubCyc and EcoCyc, with SubtiWiki
and EcoCyc being the most frequently used) provide up-todate information about these two model organisms (Caspi
et al., 2014; Keseler et al., 2013; McIntosh et al., 2012; Michna
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M. Juhas and others
et al., 2014). B. subtilis and E. coli are both endowed with
complex regulatory and metabolic networks allowing them to
thrive in a broad spectrum of environments (Buescher et al.,
2012; Kohlstedt et al., 2014; Nicolas et al., 2012; Wang et al.,
2010). B. subtilis is a commensal bacterium able to form
metabolically inactive dehydrated endospores allowing survival
in nutrient-free environments (McKenney et al., 2013). In
contrast to B. subtilis, E. coli does not form endospores and,
depending on the genome configuration, its lifestyle might
vary from commensalism to pathogenicity (Clements et al.,
2012; Leimbach et al., 2013). Some E. coli strains are important
enteric and extra-intestinal pathogens (Leimbach et al., 2013).
The E. coli strain O104 : H4 has been associated with one of the
largest recent infection outbreaks that led to more than 50
deaths and 4000 serious infections (Bielaszewska et al., 2011;
Mellmann et al., 2011; Rasko et al., 2011). A number of genes
encoding pathogenicity traits have been acquired by horizontal
gene transfer (Juhas, 2013). This may cause a significant
‘enlargement’ of the pathogenic E. coli genomes (Rasko et al.,
2008; Touchon et al., 2009). Derivatives of the B. subtilis
laboratory strain 168 and the commensal E. coli strain K-12 are
used as production platforms in biotechnology (Chen et al.,
2013; Schallmey et al., 2004). B. subtilis is an attractive host for
industrial applications due to its ability to secrete proteins into
the medium (Manabe et al., 2011, 2013; Simonen & Palva,
1993; Zweers et al., 2008). B. subtilis has been successfully
engineered for the production of riboflavin (Hao et al., 2013;
Shi et al., 2009). E. coli has been engineered for the production
of organic acids and solvents, isoprenoids, amino acids and
biofuels (Ajikumar et al., 2010; Chen et al., 2013; Clomburg &
Gonzalez, 2010, 2011; Park et al., 2012; Yim et al., 2011; Zhou
et al., 2012a, b). In many cases the development of a new
bioprocess or improvement of existing biosynthesis strategies is
hampered by lack of knowledge about each component of the
host cell chassis (Lam et al., 2012). The introduction of a novel
metabolic pathway into an existing metabolic network can lead
to unpredictable interactions (Commichau et al., 2014; Kim &
Copley, 2012). As these interactions can impair the fitness of
the engineered strain, an ideal chassis would be a wellcharacterized cell harbouring only the minimal set of functions
required to synthesize a product of interest. Systematic genome
reductions in B. subtilis and E. coli suggest that the streamlined
strains are more suitable for the production of industrially
relevant compounds (Fehér et al., 2007).
In this review we give an overview of the current
knowledge about the B. subtilis and E. coli essential genes.
Special focus is paid to the current essential gene sets and
their comparison with the original gene sets identified
10 years ago. Furthermore, we discuss recent advances in
large-scale genome engineering and genome reduction
approaches in both bacteria that aim to generate minimal
cell factories for industrial applications.
The current B. subtilis essential gene set
In 2003, a genome-scale study described 271 ORFs as
essential in B. subtilis (Kobayashi et al., 2003). This number
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was decreased to 261 as the result of experimental reevaluation over the subsequent 10 years (Akanuma et al.,
2012; Hunt et al., 2006; Mehne et al., 2013; Tanaka et al.,
2013). Of the re-evaluated set, 259 genes encoded essential
proteins, among them six of unknown function, and two
genes encoded essential RNAs (Commichau et al., 2013). In
the meantime it has been shown that four genes from the
six ORFs encoding proteins of unknown function and two
other genes are dispensable for B. subtilis growth on
complex medium (Figaro et al., 2013; Michna et al., 2014).
Recently, we found that, of the remaining two genes ylaN
and ycgG, the latter is not essential for growth of B. subtilis.
By contrast, no transformants with inactivated ylaN were
obtained (unpublished results). Moreover, our re-evaluation of the essentiality of topA encoding DNA topoisomerase I revealed that cells with inactivated topA are not viable
in LB medium (unpublished results). Thus, as of July 2014,
253 genes can be regarded as being essential in B. subtilis. The
largest group from the B. subtilis essential gene set plays a role
in protein synthesis, quality control and secretion (Fig. 1a).
Moreover, many essential genes are involved in basic
metabolism, cell wall metabolism, cell division and DNA
metabolism. A few genes encode essential proteins with
protective functions, such as RNase III, which is required to
silence phage toxins and enzymes involved in RNA turnover.
As shown above, the set of genes considered to be essential in
B. subtilis has changed substantially over the past years.
Therefore, it is plausible to assume that with the increasing
number of analyses, the current set will be further modified
in the future. The dynamics of the B. subtilis essential gene set
has been reviewed recently (Commichau et al., 2013). For upto-date information about B. subtilis essential genes we refer
to the ‘Essential genes’ page on SubtiWiki (http://subtiwiki.
uni-goettingen.de/wiki/index.php/Essential_genes).
The current E. coli essential gene set
The first E. coli minimal genome study employing genetic
footprinting led to the identification of 620 putative
essential genes in E. coli strain K-12 MG1655 (Gerdes
et al., 2003). The following systematic inactivation of nonessential ORFs by precise, single-gene deletions led to the
identification of 303 candidate essential genes in E. coli
strain K-12 BW25113 (Baba et al., 2006). Non-essential
genes in the latter study were inactivated by replacement
with the kanamycin cassette flanked by FLP recognition
target sites, thus generating in-frame deletions upon
excision of the resistance cassette (Baba et al., 2006). The
303 candidate essential genes identified correspond to 315
clusters of orthologous groups. The most abundant were
genes involved in information storage and processing,
particularly translation, ribosome structure and biogenesis
(and, to a lesser extent, transcription, DNA replication,
recombination and repair). Many genes were involved in
cell division and chromosome partitioning, cell envelope
biogenesis, post-translational modification, and lipid and
coenzyme metabolism. The function of 37 essential genes
was unknown at the time of their identification (Baba et al.,
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Microbiology 160
Escherichia coli and Bacillus subtilis essential genes
(a)
(b)
1
11 6
(c)
1013
19
17
25
12
99
41
109
5
19
67
68
70
32
59
Protein synthesis & quality control
Protective functions
Metabolism
RNA synthesis & degradation
Cell envelope & division
Unknown
DNA replication, modification & chromosome maintenance
Fig. 1. Functional categories of the B. subtilis (a) and E. coli (b) essential genes and the direct orthologues shared by both
organisms (c). The figure depicts the distribution of functional categories of the current B. subtilis and E. coli essential gene
sets. The largest proportion of essential genes is involved in protein biosynthesis, secretion and quality control, while only one
and 13 genes in B. subtilis and E. coli, respectively, are hypotheticals with unknown function. The only remaining essential gene
of unknown function in B. subtilis is ylaN, while C0614, IS128, pmrR, sokE, sokX, sroE, tpke11, yceQ, yjeV, ymiB, ynbG, yqgF
and yrbN are the 13 E. coli essential genes of unknown function. A BLASTP analysis was used to identify overlapping essential
genes shared by both organisms.
2006). A later study by Kato & Hashimoto (2007)
employing large-scale deletions to identify essential cisacting regions of the E. coli chromosome showed that 35 of
the 303 essential genes identified in 2006 were nonessential. The differences between these two essential gene
sets were probably due to strain variations (BW25141
versus MG1655), growth conditions (glucose-containing
versus glucose-free medium) and methodology (singlegene knockout versus large-scale deletions) (Kato &
Hashimoto, 2007). This study also revealed that the origin
of replication (oriC) is the only essential cis-acting DNA
sequence in the E. coli chromosome, while the terminus of
replication only affects growth, but is not essential (Kato &
Hashimoto, 2007).
Recent research advancements led to significant modifications of the original E. coli essential gene set identified
10 years ago. The current list of genes considered to be
essential in E. coli, consisting of 295 ORFs, can be found in
the EcoliWiki (http://ecoliwiki.net/colipedia/index.php/
Essential_genes). The largest proportion of the current E.
coli essential gene set (109 ORFs) is involved in protein
synthesis and quality control (Fig. 1b). Other large groups
of essential genes play roles in cell wall biosynthesis and
cell division (68 genes) and metabolism (59 genes).
Furthermore, a number of essential genes are involved in
DNA replication and chromosome maintenance and in
protective functions. A small set (10 genes) is required for
RNA synthesis and degradation, while the remaining 13
ORFs are hypotheticals with unknown function (Fig. 1b).
The functional distribution of the current E. coli essential
gene set is very similar to that of B. subtilis (Fig. 1a).
http://mic.sgmjournals.org
Inactivation of rluD encoding pseudouridine synthase was
initially shown to have a negative effect on E. coli growth
and ribosome function; however, a recent study demonstrated that rluD mutants are unstable and quickly give rise
to suppressors (O’Connor & Gregory, 2011). The rluD
suppressor mutations were found in pfrB and prfC
encoding release factor 2 (RF2) and release factor 3
(RF3), respectively (O’Connor & Gregory, 2011). The
investigation of unknown essential genes, particularly
assigning function to the short ORFs encoding essential
proteins of 50 or fewer amino acids, is challenging. Recent
analysis suggests that a number of the short essential genes
are involved in the cell’s response to a variety of stress
conditions, such as heat shock, cold shock, cell envelope
stress, oxidative stress, thiol stress and acid stress (Hemm
et al., 2010; Hobbs et al., 2010). The function of 13 ORFs of
the current E. coli essential gene set is entirely unknown
(Fig. 1b). Most of these uncharacterized ORFs are either
small RNAs (six genes) or small membrane proteinencoding genes (four genes) (Hemm et al., 2008). The
remaining three ORFs encode hypothetical proteins.
The orthologous essential genes of B. subtilis and
E. coli
To identify the set of genes essential in both B. subtilis and
E. coli we compared the corresponding protein sequences
of analysed genes from the BsubCyc and the EcoCyc
databases (Caspi et al., 2014; Keseler et al., 2013) with the
help of the BLASTP tool (Hung & Hua, 2014). The direct
orthologues of the B. subtilis and E. coli essential genes (135
ORFs) are shown in Tables S1 and S2 (available in the
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online Supplementary Material), and their assignment to
functional categories is shown in Table S3 and Fig. 1(c). Of
the B. subtilis essential genes, 58 % have direct orthologues
among E. coli essential genes, and 46 % of genes
indispensable for E. coli are essential in B. subtilis (Tables
S1–S3, Fig. 1c). The largest proportion of the orthologous
essential gene set (approx. 50 %) is involved in protein
synthesis and quality control. Another large group of direct
essential orthologues is involved in cell division and
envelope biogenesis (19 genes) (Fig. 1c). This is not
surprising as genes involved in the synthesis of proteins
and cell membrane biogenesis are considered to be part of
the ‘core’ essential gene set that is absolutely necessary for
survival of any living cell (Juhas et al., 2011). For the same
reason, B. subtilis and E. coli also share a number of genes
involved in other aspects of ‘information storage and
processing’, such as DNA replication and RNA synthesis.
Twelve of the 17 E. coli genes involved in DNA replication,
modification and chromosome maintenance are among B.
subtilis essential genes and almost all (five of six) B. subtilis
genes involved in RNA synthesis and degradation are
indispensable for E. coli (Fig. 1c). A large proportion of
shared metabolic genes could be attributed to similarities
in core metabolism and to comparable environmental
conditions (LB supplemented with glucose and growth at
37 uC), which were used to identify essential genes of both
bacteria (Baba et al., 2006; Kobayashi et al., 2003).
Variations in the sets of essential genes analysed could be
caused by the fact that different cell types might have
developed distinct solutions to the same biological problem
(Juhas et al., 2011). For example, the variations in gene sets
essential for cell membrane biosynthesis are probably due
to different architecture of the cell envelope in the Grampositive B. subtilis and Gram-negative E. coli. Genetic
background might be also the reason why B. subtilis and E.
coli do not share any of the unknown and protective
function-encoding essential genes. It remains to be elucidated why there are so many essential genes with protective
functions, particularly in E. coli. Recent analysis suggests that
a number of the short essential genes of E. coli (ythA, yobI,
yqeL, yohP, ykgR, yoaJ, ypdK, yoaK, yqcG) are involved in the
cell’s response to stress conditions, such as heat shock, cold
shock, cell envelope stress, oxidative stress, thiol stress and
acid stress (Hemm et al., 2010; Hobbs et al., 2010). Cell
envelope stress and acid stress are most relevant for the
lifestyle of the pathogenic E. coli in the human stomach and
intestine (Hobbs et al., 2010). However, it is as yet unclear
why these genes are essential for E. coli K-12 under
laboratory conditions. The above comparison shows that
while B. subtilis and E. coli share approximately 50 % of their
essential genes, there are also a number of essential genes
unique for each bacterium. This shows that the outcome of
an essential genes identification study is strongly dependent
on the bacterium cell type analysed. It is also important to
specify the strain, environmental conditions and methodology used as this might influence the outcome of the study.
For B. subtilis and E. coli essential gene analyses, strains 168
and K-12, respectively, are routinely grown at 37 uC in LB
2344
medium supplemented with glucose (Baba et al., 2006;
Kobayashi et al., 2003).
B. subtilis and E. coli genome minimization
Attempts to minimize the genomes of model bacteria such
as B. subtilis and E. coli address two major issues: (i) the
identification of the minimal gene set and the encoded
essential functions that are needed to sustain life, and (ii) the
rational design and construction of the tightly controlled cell
factories for industrially relevant products (Juhas et al.,
2012a; Nandagopal & Elowitz, 2011). Engineering bacteria
for the efficient production of a desired compound often
fails due to inhibitory interactions caused by the introduction of the heterologous pathway into an existing metabolic
network (Commichau et al., 2014; Kim & Copley, 2012;
Pitera et al., 2007). Moreover, low production levels can
result from the insufficient supply of the heterologous
enzymes with precursors (Sandmann, 2002). Thus, decreasing the number of interactions between the heterologous
pathway and the host’s metabolic network via the host’s
genome reduction could lead to more efficient bioproduction. Two approaches, bottom-up and top-down, are
employed to generate tailor-made minimal cell factories. It
is as yet unclear which of these approaches will lead to
construction of the first ‘real’ minimal cell consisting solely
of essential genes; however, based on current knowledge, the
top-down approach seems to be more straightforward.
Bottom-up genome minimization
The bottom-up approach is used to create a minimal cell
from its basic building blocks: the protein-coding essential
genes (Jewett & Forster, 2010; Juhas et al., 2012a).
Inefficient DNA synthesis and assembly methods were
among the hurdles of the bottom-up approach; however,
this has changed over recent years (Esvelt & Wang, 2013;
Fehér et al., 2012). The Gibson assembly (Gibson et al.,
2009), CPEC (circular polymerase extension cloning; Quan
& Tian, 2009; Quan & Tian, 2011), synthesis from
oligonucleotide chips (Kosuri et al., 2010; Matzas et al.,
2010), SLIC (sequence and ligase independent cloning; Li
& Elledge, 2007), SLiCE (seamless ligation cloning extract;
Zhang et al., 2012), the ligase cycling reaction (de Kok et al.,
2014) and other DNA synthesis and assembly methods
have been developed. Another hurdle was overcome when
the intact Mycoplasma mycoides genome transplanted into
Mycoplasma capricolum generated cells phenotypically
identical to the donor bacterium (Lartigue et al., 2007,
2009). This is important because genome transplantation is
required to ‘boot-up’ the synthetic genome in the genomefree recipient cell’s chassis. The synthetic 1.08 Mbp M.
mycoides genome dubbed ‘JCVI-syn1.0’ assembled in yeast
was successfully transplanted into M. capricolum, thus
generating cells with the M. mycoides phenotype (Gibson
et al., 2009, 2010; Itaya, 2010). Recent synthesis of a whole
functional yeast chromosome (Annaluru et al., 2014)
suggests that synthetic biology has reached the stage where
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Escherichia coli and Bacillus subtilis essential genes
tailor-made prokaryotic and eukaryotic organisms can be
constructed from standardized biological parts. However,
this is still far from the case. The minimal set of genes
required to sustain life is not yet known. Even the essential
gene sets of the two well-studied model organisms B.
subtilis and E. coli, modified significantly over the last
10 years, are still undergoing alterations. Thus, based on
current knowledge, it is hard to imagine that essential
genes from different sources could be put together and
transplanted into a cell chassis to obtain a functional
minimal cell factory. A conservative approach, including
more genes than those constituting an absolutely minimal
gene set, could make this goal more realistic. The genome
of such a cell could be subsequently further minimized by
the top-down approach. Alternatively, a straightaway topdown approach followed by the genome engineering-based
augmentation of the reduced cell with desired properties
could lead to the construction of the minimal cell factory.
Top-down genome minimization
The top-down approach is used to streamline existing cells
by deleting large non-essential parts of their genomes. As
the B. subtilis and E. coli genome sequences are available
(Blattner et al., 1997; Kunst et al., 1997) they were often
targets of top-down minimization. Significant progress has
been made in the genome reduction of both model bacteria
in recent years (Fehér et al., 2007) (Fig. 2). The first
streamlined B. subtilis strain lacking 7.7 % of the genome
was generated by deleting the prophage PBSX, the three
prophage-like elements skin, prow1 and prow3 and the pks
gene cluster, in a strain that was already cured from the
cryptic prophage SPb (Dorenbos et al., 2002; Westers et al.,
2003). Interestingly, the loss of 332 genes (about 325 kbp)
did not cause a growth defect, and the flux through central
carbon metabolism was identical to that of the parental
strain. Moreover, although the reduced-genome strain was
more motile than the parent strain, their competence and
sporulation phenotypes were comparable. This showed
that regions of the B. subtilis chromosome can be deleted
without affecting viability under laboratory growth conditions. It is unclear why these DNA regions reside in the
bacterial genome; their selective advantage has yet to be
identified. In another work, based on the genome-scale
identification of essential genes (Kobayashi et al., 2003), B.
subtilis lacking 17 dispensable genomic regions, in addition
to the prophage-like elements skin, prow1–6, the cryptic
prophage SPb, the prophage PBSX and the pks gene cluster,
was constructed (Ara et al., 2007). The resulting strain with
a 3.2 Mbp genome was viable but showed unstable growth
rate, cell morphology and protein production after extensive
culturing (Ara et al., 2007). In 2008, the construction of the
reduced-genome B. subtilis strain MGB874 lacking 874 kbp
(20.7 %) of genomic DNA was reported (Morimoto et al.,
2008). In this study, the impact of each large genomic
deletion on growth was tested prior to combining all the
deletions in a single strain. In total, 11 dispensable regions
with lengths between 11 and 195 kbp were sequentially
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deleted from the parent strain MGB469. Compared with the
parent strain, the constructed B. subtilis MGB874 had a similar
growth rate and yield in LB medium, while showing only a
slightly reduced growth in minimal medium (Morimoto et al.,
2008). Transcriptome analyses revealed that the transcriptional regulatory network in the reduced-genome strain was
disturbed during exponential growth and that it was
reorganized after entry into the stationary growth phase
(Morimoto et al., 2008). Although the molecular basis for this
phenomenon remains to be elucidated, strain MGB874 serves
as a cell factory for the production of proteins and other
valuable compounds and as the promising basis for further
genome reduction (see below). Recently, a B. subtilis strain
lacking 35 % of the genome was constructed by sequentially
combining deletions from the repertoire of the chromosome
regions dispensable for growth (Tanaka et al., 2013).
Like B. subtilis, E. coli has been also subjected to systematic
genome reduction. In 2002, the first genome-reduced E.
coli strain, MDS12 lacking 8.1 % of the genome, was
constructed by deleting 12 strain-specific genomic islands
(K-islands) from E. coli K-12 without leaving scars or
markers (Kolisnychenko et al., 2002) (Fig. 2). The doubling
time of MGB469 in LB and minimal medium with and
without Casamino acids was similar to that of the parent
strain. Surprisingly, the reduced-genome strain reached
about 10 % higher optical density in all tested media, thus
showing that deletion of K-islands provides bacteria with a
selective growth advantage under the tested conditions. In
the same year, Yu et al. (2002) reported E. coli genome
reduction by 6.7 % using the Tn5 transposon-targeted Cre/
loxP excision system. The Cre/loxP excision system method
is very elegant because it allows deletion of almost any
non-essential E. coli DNA (Yu et al., 2002). First, two
independent transposon libraries harbouring loxP recombination sites are generated and the transposon insertion
sites are identified by sequencing. Next, selected loxP sites
are combined in a single strain by transduction. Finally, the
genomic regions located between the two loxP sites are
excised from the chromosome by activation of the Cre
recombinase. As in the previous study, the genome-reduced
E. coli with four large-scale deletions exhibited normal
growth in LB medium. A few years later, Hashimoto et al.
(2005) reported the construction of a series of E. coli
mutants with marker-less deletions between 2.4 and 29 % of
the genome. Although the strain with the largest deletion
was viable in the rich medium, its nucleoid and cell
morphology were different. Several small nucleoids were
localized peripherally close to the cell envelope. Moreover,
the cells of the deletion mutants were wider and longer than
those of the parent strains. This study revealed that large
parts of the E. coli genome can be deleted without a negative
effect on growth. To obtain a stable and robust growing
reduced-genome E. coli strain, Pósfai et al. (2006) first
identified non-essential genome parts, such as mobile and
cryptic virulence genes. Next, individual large-scale, scarless
deletions were made and growth of the resulting strains was
tested on minimal medium. Finally, deletions not affecting
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M. Juhas and others
Bacillus subtilis 168
20
14
20
03
Essential gene pairs
(Thomaides et al.)
20
13
Δ25%
(Ara et al.)
Re-evaluation of
essential genes
(Commichau et al.
& Michna et al.)
20
12
Essential genes
(Kobayashi et al.)
20
08
Genome sequence
(Kunst et al.)
Δ20.7%
(Morimoto et al.)
20
07
Δ7.7%
(Westers et al.)
19
97
Two subgenomes
(ltaya & Tanaka)
146 individual deletions
(Tanaka et al.)
Escherichia coli K-12
Re-evaluation of
essential genes
(Baba et al.)
08
20
07
20
06
20
05
20
03
20
02
Δ29.7%
(Hashimoto et al.)
Δ35.2%
(Hirokawa et al.)
Two linear
genomes
(Liang et al.)
Δ15.3%
(Pósfai et al.)
Δ8.1%
(Kolisnychenko et al.)
20
19
97
Genome sequence
(Blattner et al.)
Δ22%
(Mizoguchi et al.)
13
Δ6.7%
(Yu et al.)
20
Essential genes
(Gerdes et al.)
Δ30%
(Kato & Hashimoto)
Fig. 2. Genome-scale engineering in B. subtilis and E. coli. Timeline showing the major achievements in the large-scale
engineering of the B. subtilis and E. coli genomes.
growth were serially combined in a single strain resulting in
the removal of 15.3 % of the chromosome. Besides confirming that bacteria with a considerably reduced genome can be
constructed in the laboratory, this study also showed that the
reduced-genome strains are endowed with beneficial properties such as increased electroporation efficiencies and
decreased mutation rates (see below) (Pósfai et al., 2006).
Two studies by Mizoguchi et al. (2007, 2008) and Kato &
Hashimoto (2007, 2008) removed 22 and 30 % of the E. coli
genome, respectively. The regions targeted for deletion were
selected by comparative genomics between E. coli and the
natural reduced-genome symbiotic bacteria Buchnera spp.
Before combination of deletions in a single strain each
intermediate deletion strain was characterized to maintain
robust growth in minimal medium. Similar to the work of
Pósfai et al. (2006) the resulting streamlined E. coli strain
MGF-01 lacking 22 % of the genome was endowed with
emerging properties. Furthermore, strain MGF-01 did not
exhibit any growth defects in the exponential phase and grew
2346
to a 1.5 times higher final cell density than the wild-type in
M9 minimal medium, which is a trait beneficial for
biotechnology applications (see below) (Mizoguchi et al.,
2008). Recently, two reduced-genome E. coli K-12 strains
DGF-327 and DGF-298 lacking 29 and 35.2 % of the
chromosome, respectively, were constructed (Hirokawa
et al., 2013). Interestingly, during the construction of strains
DGF-327 and DGF-298 bacteria accumulated mutations in
the rph and ilvG1 genes encoding RNase PH and the large
subunit of the acetohydroxybutanoate synthase, respectively.
These mutations are beneficial because they enable sufficient
production of pyrimidine and isoleucine needed for growth
in minimal medium (Jensen, 1993; Lawther et al., 1981). It is
tempting to assume that the frameshift mutations in rph and
ilvG1 had accumulated during the ‘domestication’ of E. coli
K-12 (Blattner et al., 1997). The ilvG1 mutation disrupts one
of the three biosynthetic pathways for isoleucine and valine
while the mutation at the end of rph reduces expression of
the downstream pyrE, which in turn increases the demand
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Microbiology 160
Escherichia coli and Bacillus subtilis essential genes
for pyrimidine. Thus, the cryptic genetic information can be
recovered in the reduced-genome bacteria by the accumulation of spontaneous mutations and selection for faster
growing isolates. The step-wise genome reduction and
evolution of the reduced-genome bacteria for robust growth
might be an elegant strategy to promote genome minimization (see below).
Taken together, several studies showed that large regions of
the E. coli and B. subtilis genomes are dispensable under
standard laboratory growth conditions. However, to further
reduce the genomes of these two model organisms it is
crucial that we understand the functions of uncharacterized
genes and the functional and genetic interactions between
essential genes (both known and unknown) in the cell. This
knowledge could prevent genetic and phenotypic instabilities caused by severe genome surgeries during the genome
streamlining process. Partially this lack of knowledge can
be replenished in the process of genome minimization.
Moreover, better understanding of cellular processes is also
necessary for advancements in the bottom-up construction
of E. coli- and B. subtilis-based minimal cells.
Combining genome reduction and evolution for
the construction of fast-growing minimal cells
Besides numerous beneficial traits (Manabe et al., 2013; Pósfai
et al., 2006), genome reductions in B. subtilis and E. coli often
led to unwanted phenotypes, such as growth defects, and
aberrant cell and nucleoid morphologies (Hashimoto et al.,
2005). Thus, a knowledge-driven approach should be used
to minimize the risk that B. subtilis and E. coli genome
reductions end up in a ‘blind alley’. The genomic regions to
be deleted should be carefully selected based on current
information about the target genes stored in the communitycurated databases SubtiWiki and EcoliWiki (McIntosh et al.,
2012; Michna et al., 2014). Even if the target gene was not
designated essential, its inactivation can cause a severe growth
defect. For instance, deletion of rocG encoding the glutamate
dehydrogenase RocG severely affects growth of B. subtilis in
complex medium (Gunka et al., 2013). This is not surprising
as RocG is a bifunctional enzyme active both in glutamate
catabolism and in controlling DNA-binding activity of the
transcription factor GltC (Commichau et al., 2007). In the
absence of RocG, GltC activates the expression of gltA and
gltB involved in glutamate biosynthesis, which is not required
on complex medium. Simultaneous uptake of glutamate from
the medium and blocking its catabolism might increase
glutamate concentration to toxic levels. Furthermore, the B.
subtilis and E. coli genomes may contain genes encoding
enzymes with redundant activities. For instance, the reducedgenome B. subtilis should synthesize at least one enzyme
producing the recently discovered essential second messenger
c-di-AMP (Corrigan & Gründling, 2013; Mehne et al., 2013).
Moreover, multiple rRNA operons were shown to be essential
for the efficient growth of B. subtilis (Yano et al., 2013). Thus,
each genomic region targeted for deletion and the order of
deletions has to be considered carefully as strains with severe
http://mic.sgmjournals.org
growth defects do not provide a suitable basis for further
genome reduction. Once the reduced-genome strain retaining
robust growth is obtained it can be used for the next
sequential deletions. In the ideal case, the deletions can be
made until construction of the ultimate reduced-genome
strain (Fig. 3). However, it is plausible to assume that several
deletions will lead to growth defects. Therefore, the isolation
of robustly growing strains by continuous selection under the
desired conditions might identify suppressor mutants with
enhanced growth rates. For instance, mutations in rpoC,
an essential E. coli gene encoding the b9 subunit of RNA
polymerase, were shown to increase the growth rate of E. coli
K-12 MG1655 in glycerol M9 minimal medium by 60 %.
Furthermore, rpoC mutants converted the carbon source to
biomass up to 35 % more efficiently than the wild-type strain
(Conrad et al., 2010). These phenotypes resulted from the
massive adaptive rewiring of the E. coli transcription network
via extensive changes in the gene expression profile and
reprogramming of the kinetic parameters of RNA polymerase
(Conrad et al., 2010). The identification of growth defectrelieving mutations by whole-genome sequencing might
uncover new insights into gene functions, interactions and
moonlighting (Copley, 2012) (Fig. 3). It was shown that the
‘decryptification’ of B. subtilis and E. coli genetic information
by spontaneous mutagenesis during growth under selection
can recover growth defects of reduced-genome strains
(Hirokawa et al., 2013). Moreover, overexpression of nonessential genes can completely compensate for the loss of
essential genes (Bergmiller et al., 2012). Thus, each deletion
strain has to be carefully analysed and tested for the presence
of growth defect-restoring suppressor mutations. Once an
‘extremely’ reduced-genome strain has been isolated, the
remaining non-essential genes can be identified by saturated
transposon mutagenesis. Finally, the identified dispensable
genomic regions can be removed employing the tools for
marker-free gene deletions (Fig. 3).
Minimal cell factories
B. subtilis and E. coli genome reductions often led to
beneficial traits (Manabe et al., 2011, 2013; Mizoguchi
et al., 2008). B. subtilis strain MGB874 lacking 20.7 % of
the genome produced significantly higher levels of the
heterologous enzymes alkaline cellulase and protease than
the parent strain (Morimoto et al., 2008). The production
of extracellular enzymes by B. subtilis MGB874 was further
enhanced by deleting genes from the arginine degradation
pathway (Manabe et al., 2011, 2013). Reductions of the E.
coli genome also led to beneficial properties. E. coli strains
without redundant metabolic pathways produced higher
quantities of ethanol from hexose and pentose sugars
(Trinh et al., 2008). E. coli lacking 22 % of the genomic
DNA produced 2.4-fold more threonine that the wild-type
strain (Mizoguchi et al., 2008). The E. coli strains MDS41–
43 generated by deleting up to 15.3 % of the dispensable
genomic DNA, such as insertion sequences, exhibited
numerous beneficial traits, for example a decreased mutation rate and increased electroporation efficiency (Pósfai
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2347
M. Juhas and others
Progenitor
strain
Deletion of
genomic regions
Characterization
of evolved strains
Robust
growth
Evolution of reducedgenome strains
Growth
defect
Progenitor
mini cell
Transposon mutagenesis
to identify non-essential
genomic regions
‘chassis’. The outcome of an essential genes identification
study is strongly influenced by growth conditions and the
genetic background of the investigated cell. As described
above, B. subtilis and E. coli share approximately 50 % of
their essential genes. Currently, there is only one essential
gene of unknown function in B. subtilis, while the functions
of 13 essential ORFs are uncharacterized in E. coli. In the
past years, B. subtilis and E. coli were subjected to genome
reductions that often led to unexpected beneficial traits,
while not having negative impacts on fitness. On the basis
of our current knowledge about essential genes we are yet
unable to create tailor-made minimal cells for industrial
applications encoded by genomes composed of nothing
else but essential genes. However, advances highlighted in
this review suggest that the minimized cell generated from
B. subtilis and E. coli could be used as a suitable chassis for
biotechnology applications. It will be very interesting to see
which organism provides the first chassis for the true
minimal cell factory.
Acknowledgements
Removal of residual
non-essential
genomic regions
Mini cell
Fig. 3. Construction of the minimal cell factories. The figure shows
the iterative cycle for the generation of cell factories with
minimized/reduced genomes.
Cordial thanks to all researchers who contributed to advancements
in the analysis of E. coli and B. subtilis essential genes and to
investigation of minimal genomes in general. Work in the authors’
laboratories is supported by the DFG (grant CO 1139/1-1), the
Fonds der Chemischen Industrie (http://www.vci.de/fonds) and the
Göttingen Centre for Molecular Biology (GZMB). D. R. R. was
supported by the Göttingen Graduate School for Neuroscience and
Molecular Biosciences (DFG grant GSC 226/2). The authors declare
no conflicts of interest associated with this paper.
References
et al., 2006). Strain-specific genes targeted for deletion in
this study were chosen by comparative genomics between six
E. coli genomes (Pósfai et al., 2006). The decreased mutation
rate was attributed to the removal of insertion sequence
elements that can spontaneously ‘jump’ between genes, thus
interfering with the encoded functions. The reducedgenome E. coli strain MDS42 was further metabolically
engineered to improve the production of threonine by
overexpressing feedback-resistant biosynthetic enzymes, by
deleting the native threonine dehydrogenase and threonine
transporter genes and by incorporating a mutant threonine
exporter gene (Lee et al., 2009). Strikingly, the resulting
strain MDS-205 produced 83 % more L-threonine than the
wild-type. This demonstrates that lowering metabolic
burden and increasing metabolic efficiency by deleting
non-essential genes can generate industrially relevant
production strains (Lee et al., 2009).
Isoprenoid pathway optimization for Taxol precursor overproduction
in Escherichia coli. Science 330, 70–74.
Akanuma, G., Nanamiya, H., Natori, Y., Yano, K., Suzuki, S., Omata,
S., Ishizuka, M., Sekine, Y. & Kawamura, F. (2012). Inactivation of
ribosomal protein genes in Bacillus subtilis reveals importance of each
ribosomal protein for cell proliferation and cell differentiation.
J Bacteriol 194, 6282–6291.
Annaluru, N., Muller, H., Mitchell, L. A., Ramalingam, S.,
Stracquadanio, G., Richardson, S. M., Dymond, J. S., Kuang, Z.,
Scheifele, L. Z. & other authors (2014). Total synthesis of a
functional designer eukaryotic chromosome. Science 344, 55–58.
Ara, K., Ozaki, K., Nakamura, K., Yamane, K., Sekiguchi, J. &
Ogasawara, N. (2007). Bacillus minimum genome factory: effective
utilization of microbial genome information. Biotechnol Appl Biochem
46, 169–178.
Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M.,
Datsenko, K. A., Tomita, M., Wanner, B. L. & Mori, H. (2006).
Construction of Escherichia coli K-12 in-frame, single-gene knockout
mutants: the Keio collection. Mol Syst Biol 2, 2006.0008.
Conclusions
B. subtilis and E. coli are both well-studied model bacteria
that serve as hosts for a plethora of biotechnology
applications. Determination of their minimal genomes will
lead to the construction of the more suitable cellular
2348
Ajikumar, P. K., Xiao, W. H., Tyo, K. E., Wang, Y., Simeon, F., Leonard,
E., Mucha, O., Phon, T. H., Pfeifer, B. & Stephanopoulos, G. (2010).
Bergmiller, T., Ackermann, M. & Silander, O. K. (2012). Patterns of
evolutionary conservation of essential genes correlate with their
compensability. PLoS Genet 8, e1002803.
Bielaszewska, M., Mellmann, A., Zhang, W., Köck, R., Fruth, A.,
Bauwens, A., Peters, G. & Karch, H. (2011). Characterisation of the
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 13:52:43
Microbiology 160
Escherichia coli and Bacillus subtilis essential genes
Escherichia coli strain associated with an outbreak of haemolytic
uraemic syndrome in Germany, 2011: a microbiological study. Lancet
Infect Dis 11, 671–676.
de Kok, S., Stanton, L. H., Slaby, T. & other authors (2014). Rapid
Blattner, F. R., Plunkett, G., Bloch, C. A. & other authors (1997). The
Dorenbos, R., Stein, T., Kabel, J., Bruand, C., Bolhuis, A., Bron, S.,
Quax, W. J. & Van Dijl, J. M. (2002). Thiol-disulfide oxidoreductases
complete genome sequence of Escherichia coli K-12. Science 277,
1453–1462.
and reliable DNA assembly via ligase cycling reaction. ACS Synth Biol
3, 97–106.
Boutros, M. & Ahringer, J. (2008). The art and design of genetic
are essential for the production of the lantibiotic sublancin 168. J Biol
Chem 277, 16682–16688.
screens: RNA interference. Nat Rev Genet 9, 554–566.
Durand, S., Gilet, L. & Condon, C. (2012). The essential function of B.
Buescher, J. M., Liebermeister, W., Jules, M., Uhr, M., Muntel, J.,
Botella, E., Hessling, B., Kleijn, R. J., Le Chat, L. & other authors
(2012). Global network reorganization during dynamic adaptations of
subtilis RNase III is to silence foreign toxin genes. PLoS Genet 8,
e1003181.
Bacillus subtilis metabolism. Science 335, 1099–1103.
Esvelt, K. M. & Wang, H. H. (2013). Genome-scale engineering for
systems and synthetic biology. Mol Syst Biol 9, 641.
Bumann, D. (2008). Has nature already identified all useful
Fehér, T., Papp, B., Pal, C. & Pósfai, G. (2007). Systematic genome
antibacterial targets? Curr Opin Microbiol 11, 387–392.
reductions: theoretical and experimental approaches. Chem Rev 107,
3498–3513.
Caspi, R., Altman, T., Billington, R., Dreher, K., Foerster, H., Fulcher,
C. A., Holland, T. A., Keseler, I. M., Kothari, A. & other authors (2014).
The MetaCyc database of metabolic pathways and enzymes and the
BioCyc collection of Pathway/Genome Databases. Nucleic Acids Res
42 (D1), D459–D471.
Chen, X., Zhou, L., Tian, K., Kumar, A., Singh, S., Prior, B. A. & Wang,
Z. (2013). Metabolic engineering of Escherichia coli: a sustainable
industrial platform for bio-based chemical production. Biotechnol
Adv 31, 1200–1223.
Christen, B., Abeliuk, E., Collier, J. M., Kalogeraki, V. S., Passarelli,
B., Coller, J. A., Fero, M. J., McAdams, H. H. & Shapiro, L. (2011). The
essential genome of a bacterium. Mol Syst Biol 7, 528.
Clements, A., Young, J. C., Constantinou, N. & Frankel, G. (2012).
Infection strategies of enteric pathogenic Escherichia coli. Gut
Microbes 3, 71–87.
Fehér, T., Burland, V. & Pósfai, G. (2012). In the fast lane: large-scale
bacterial genome engineering. J Biotechnol 160, 72–79.
Figaro, S., Durand, S., Gilet, L., Cayet, N., Sachse, M. & Condon, C.
(2013). Bacillus subtilis mutants with knockouts of the genes encoding
ribonucleases RNase Y and RNase J1 are viable, with major defects in
cell morphology, sporulation, and competence. J Bacteriol 195, 2340–
2348.
French, C. T., Lao, P., Loraine, A. E., Matthews, B. T., Yu, H. & Dybvig,
K. (2008). Large-scale transposon mutagenesis of Mycoplasma
pulmonis. Mol Microbiol 69, 67–76.
Gerdes, S., Scholle, M., Campbell, J., Balazsi, G., Ravasz, E.,
Daugherty, M. D., Somera, A. L., Kyrpides, N. C., Anderson, I. & other
authors (2003). Experimental determination and system level analysis
Clomburg, J. M. & Gonzalez, R. (2010). Biofuel production in
of essential genes in Escherichia coli MG1655. J Bacteriol 185, 5673–
5684.
Escherichia coli: the role of metabolic engineering and synthetic
biology. Appl Microbiol Biotechnol 86, 419–434.
Gibson, D., Young, L., Chuang, R., Venter, J., Hutchison, C. & Smith,
H. (2009). Enzymatic assembly of DNA molecules up to several
Clomburg, J. M. & Gonzalez, R. (2011). Metabolic engineering of
hundred kilobases. Nat Methods 6, 343–345.
Escherichia coli for the production of 1,2-propanediol from glycerol.
Biotechnol Bioeng 108, 867–879.
Gibson, D., Glass, J., Lartigue, C., Noskov, V. N., Chuang, R.-Y.,
Algire, M. A., Benders, G. A., Montague, M. G., Ma, L. & other authors
(2010). Creation of a bacterial cell controlled by a chemically
Commichau, F. M., Herzberg, C., Tripal, P., Valerius, O. & Stülke, J.
(2007). A regulatory protein–protein interaction governs glutamate
biosynthesis in Bacillus subtilis: the glutamate dehydrogenase RocG
moonlights in controlling the transcription factor GltC. Mol Microbiol
65, 642–654.
Commichau, F. M., Pietack, N. & Stülke, J. (2013). Essential genes in
Bacillus subtilis: a re-evaluation after ten years. Mol Biosyst 9, 1068–
1075.
Commichau, F. M., Alzinger, A., Sande, R., Bretzel, W., Meyer, F. M.,
Chevreux, B., Wyss, M., Hohmann, H. P. & Prágai, Z. (2014).
Overexpression of a non-native deoxyxylulose-dependent vitamin B6
pathway in Bacillus subtilis for the production of pyridoxine. Metab
Eng 25, 38–49.
Conrad, T. M., Frazier, M., Joyce, A. R., Cho, B. K., Knight, E. M.,
Lewis, N. E., Landick, R. & Palsson, B. (2010). RNA polymerase
mutants found through adaptive evolution reprogram Escherichia coli
for optimal growth in minimal media. Proc Natl Acad Sci U S A 107,
20500–20505.
Copley, S. D. (2012). Moonlighting is mainstream: paradigm
adjustment required. Bioessays 34, 578–588.
synthesized genome. Science 329, 52–56.
Gunka, K., Stannek, L., Care, R. A. & Commichau, F. M. (2013).
Selection-driven accumulation of suppressor mutants in Bacillus
subtilis: the apparent high mutation frequency of the cryptic gudB
gene and the rapid clonal expansion of gudB+ suppressors are due to
growth under selection. PLoS ONE 8, e66120.
Hao, T., Han, B., Ma, H., Fu, J., Wang, H., Wang, Z., Tang, B., Chen, T.
& Zhao, X. (2013). In silico metabolic engineering of Bacillus subtilis
for improved production of riboflavin, Egl-237, (R,R)-2,3-butanediol
and isobutanol. Mol Biosyst 9, 2034–2044.
Hashimoto, M., Ichimura, T., Mizoguchi, H., Tanaka, K., Fujimitsu, K.,
Keyamura, K., Ote, T., Yamakawa, T., Yamazaki, Y. & other authors
(2005). Cell size and nucleoid organization of engineered Escherichia
coli cells with a reduced genome. Mol Microbiol 55, 137–149.
Hemm, M. R., Paul, B. J., Schneider, T. D., Storz, G. & Rudd, K. E.
(2008). Small membrane proteins found by comparative genomics
and ribosome binding site models. Mol Microbiol 70, 1487–1501.
Hemm, M. R., Paul, B. J., Miranda-Rı́os, J., Zhang, A., Soltanzad, N. &
Storz, G. (2010). Small stress response proteins in Escherichia coli:
second messenger enters the fray. Nat Rev Microbiol 11, 513–524.
proteins missed by classical proteomic studies. J Bacteriol 192, 46–
58.
de Berardinis, V., Vallenet, D., Castelli, V., Besnard, M., Pinet, A.,
Cruaud, C., Samair, S., Lechaplais, C., Gyapay, G. & other authors
(2008). A complete collection of single-gene deletion mutants of
Hirokawa, Y., Kawano, H., Tanaka-Masuda, K., Nakamura, N.,
Nakagawa, A., Ito, M., Mori, H., Oshima, T. & Ogasawara, N.
(2013). Genetic manipulations restored the growth fitness of reduced-
Acinetobacter baylyi ADP1. Mol Syst Biol 4, 174.
genome Escherichia coli. J Biosci Bioeng 116, 52–58.
Corrigan, R. M. & Gründling, A. (2013). Cyclic di-AMP: another
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 13:52:43
2349
M. Juhas and others
Hobbs, E. C., Astarita, J. L. & Storz, G. (2010). Small RNAs and small
proteins involved in resistance to cell envelope stress and acid shock
in Escherichia coli: analysis of a bar-coded mutant collection.
J Bacteriol 192, 59–67.
Hung, C. L. & Hua, G. J. (2014). Local alignment tool based on
Hadoop framework and GPU architecture. Biomed Res Int 2014,
541490.
Hunt, A., Rawlins, J. P., Thomaides, H. B. & Errington, J. (2006).
Functional analysis of 11 putative essential genes in Bacillus subtilis.
Microbiology 152, 2895–2907.
authors (1997). The complete genome sequence of the gram-positive
bacterium Bacillus subtilis. Nature 390, 249–256.
Lam, C. M., Suárez Diez, M., Godinho, M. & Martins dos Santos, V. A.
(2012). Programmable bacterial catalysis – designing cells for
biosynthesis of value-added compounds. FEBS Lett 586, 2184–2190.
Langridge, G. C., Phan, M. D., Turner, D. J., Perkins, T. T., Parts, L.,
Haase, J., Charles, I., Maskell, D. J., Peters, S. E. & other authors
(2009). Simultaneous assay of every Salmonella typhi gene using one
million transposon mutants. Genome Res 19, 2308–2316.
Itaya, M. (2010). A synthetic DNA transplant. Nat Biotechnol 28, 687–689.
Lartigue, C., Glass, J., Alperovich, N., Pieper, R., Parmar, P.,
Hutchison, C., Smith, H. & Venter, J. (2007). Genome transplantation
Jensen, K. F. (1993). The Escherichia coli K-12 ‘‘wild types’’ W3110
and MG1655 have an rph frameshift mutation that leads to
pyrimidine starvation due to low pyrE expression levels. J Bacteriol
175, 3401–3407.
Lartigue, C., Vashee, S., Algire, M., Chuang, R.-Y., Benders, G. A.,
Ma, L., Noskov, V. N., Denisova, E. A., Gibson, D. G. & other authors
(2009). Creating bacterial strains from genomes that have been cloned
Jewett, M. C. & Forster, A. C. (2010). Update on designing and
and engineered in yeast. Science 325, 1693–1696.
building minimal cells. Curr Opin Biotechnol 21, 697–703.
Lawther, R. P., Calhoun, D. H., Adams, C. W., Hauser, C. A., Gray, J. &
Hatfield, G. W. (1981). Molecular basis of valine resistance in
Juhas, M. (2013). Horizontal gene transfer in human pathogens. Crit
Rev Microbiol 1–8. doi:10.3109/1040841X.2013.804031 [Epub ahead of
print]
Juhas, M., Eberl, L. & Glass, J. I. (2011). Essence of life: essential genes
of minimal genomes. Trends Cell Biol 21, 562–568.
Juhas, M., Eberl, L. & Church, G. M. (2012a). Essential genes as
antimicrobial targets and cornerstones of synthetic biology. Trends
Biotechnol 30, 601–607.
Juhas, M., Stark, M., von Mering, C., Lumjiaktase, P., Crook, D. W.,
Valvano, M. A. & Eberl, L. (2012b). High confidence prediction of
essential genes in Burkholderia cenocepacia. PLoS ONE 7, e40064.
Juhas, M., Davenport, P. W., Brown, J. R., Yarkoni, O. & Ajioka, J. W.
(2013). Meeting report: The Cambridge BioDesign TechEvent –
Synthetic Biology, a new ‘‘Age of Wonder’’? Biotechnol J 8, 761–763.
in bacteria: changing one species to another. Science 317, 632–638.
Escherichia coli K-12. Proc Natl Acad Sci U S A 78, 922–925.
Lee, J. H., Sung, B. H., Kim, M. S., Blattner, F. R., Yoon, B. H., Kim,
J. H. & Kim, S. C. (2009). Metabolic engineering of a reduced-genome
strain of Escherichia coli for L-threonine production. Microb Cell Fact
8, 2.
Leimbach, A., Hacker, J. & Dobrindt, U. (2013). E. coli as an all-
rounder: the thin line between commensalism and pathogenicity.
Curr Top Microbiol Immunol 358, 3–32.
Li, M. Z. & Elledge, S. J. (2007). Harnessing homologous
recombination in vitro to generate recombinant DNA via SLIC. Nat
Methods 4, 251–256.
Manabe, K., Kageyama, Y., Morimoto, T., Ozawa, T., Sawada, K.,
Endo, K., Tohata, M., Ara, K., Ozaki, K. & Ogasawara, N. (2011).
Kato, J. & Hashimoto, M. (2008). Construction of long chromosomal
Combined effect of improved cell yield and increased specific
productivity enhances recombinant enzyme production in genomereduced Bacillus subtilis strain MGB874. Appl Environ Microbiol 77,
8370–8381.
deletion mutants of Escherichia coli and minimization of the genome.
Methods Mol Biol 416, 279–293.
Manabe, K., Kageyama, Y., Morimoto, T., Shimizu, E., Takahashi, H.,
Kanaya, S., Ara, K., Ozaki, K. & Ogasawara, N. (2013). Improved
Keseler, I. M., Mackie, A., Peralta-Gil, M., Santos-Zavaleta, A., GamaCastro, S., Bonavides-Martinez, C., Fulcher, C., Huerta, A. M., Kothari,
A. & other authors (2013). EcoCyc: fusing model organism databases
production of secreted heterologous enzyme in Bacillus subtilis strain
MGB874 via modification of glutamate metabolism and growth
conditions. Microb Cell Fact 12, 18.
with systems biology. Nucleic Acids Res 41 (D1), D605–D612.
Matzas, M., Stähler, P. F., Kefer, N., Siebelt, N., Boisguérin, V.,
Leonard, J. T., Keller, A., Stähler, C. F., Häberle, P. & other authors
(2010). High-fidelity gene synthesis by retrieval of sequence-verified
Kato, J. & Hashimoto, M. (2007). Construction of consecutive
deletions of the Escherichia coli chromosome. Mol Syst Biol 3, 132.
Kim, J. & Copley, S. D. (2012). Inhibitory cross-talk upon introduc-
tion of a new metabolic pathway into an existing metabolic network.
Proc Natl Acad Sci U S A 109, E2856–E2864.
Kobayashi, K., Ehrlich, S., Albertini, A., Amati, G., Andersen, K. K.,
Arnaud, M., Asai, K., Ashikaga, S., Aymerich, S. & other authors
(2003). Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A 100,
4678–4683.
Kohlstedt, M., Sappa, P. K., Meyer, H., Maaß, S., Zaprasis, A.,
Hoffmann, T., Becker, J., Steil, L., Hecker, M. & other authors (2014).
Adaptation of Bacillus subtilis carbon core metabolism to simultaneous nutrient limitation and osmotic challenge: a multi-omics
perspective. Environ Microbiol 16, 1898–1917.
DNA identified using high-throughput
Biotechnol 28, 1291–1294.
pyrosequencing.
Nat
McCutcheon, J. P. & Moran, N. A. (2010). Functional convergence in
reduced genomes of bacterial symbionts spanning 200 My of
evolution. Genome Biol Evol 2, 708–718.
McIntosh, B. K., Renfro, D. P., Knapp, G. S., Lairikyengbam, C. R.,
Liles, N. M., Niu, L., Supak, A. M., Venkatraman, A., Zweifel, A. E. &
other authors (2012). EcoliWiki: a wiki-based community resource
for Escherichia coli. Nucleic Acids Res 40 (D1), D1270–D1277.
McKenney, P. T., Driks, A. & Eichenberger, P. (2013). The Bacillus
Kolisnychenko, V., Plunkett, G., Herring, C. D., Fehér, T., Pósfai, J.,
Blattner, F. R. & Pósfai, G. (2002). Engineering a reduced Escherichia
subtilis endospore: assembly and functions of the multilayered coat.
Nat Rev Microbiol 11, 33–44.
coli genome. Genome Res 12, 640–647.
Mehne, F. M., Gunka, K., Eilers, H., Herzberg, C., Kaever, V. & Stülke,
J. (2013). Cyclic di-AMP homeostasis in Bacillus subtilis: both lack
Kosuri, S., Eroshenko, N., Leproust, E. M., Super, M., Way, J., Li, J. B.
& Church, G. M. (2010). Scalable gene synthesis by selective
amplification of DNA pools from high-fidelity microchips. Nat
Biotechnol 28, 1295–1299.
Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G.,
Azevedo, V., Bertero, M. G., Bessières, P., Bolotin, A. & other
2350
and high level accumulation of the nucleotide are detrimental for cell
growth. J Biol Chem 288, 2004–2017.
Mellmann, A., Harmsen, D., Cummings, C. A., Zentz, E. B., Leopold,
S. R., Rico, A., Prior, K., Szczepanowski, R., Ji, Y. & other authors
(2011). Prospective genomic characterization of the German
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 13:52:43
Microbiology 160
Escherichia coli and Bacillus subtilis essential genes
enterohemorrhagic Escherichia coli O104 : H4 outbreak by rapid next
generation sequencing technology. PLoS ONE 6, e22751.
Seo, S. W., Yang, J., Min, B. E., Jang, S., Lim, J. H., Lim, H. G., Kim,
S. C., Kim, S. Y., Jeong, J. H. & Jung, G. Y. (2013). Synthetic biology:
Michna, R. H., Commichau, F. M., Tödter, D., Zschiedrich, C. P. &
Stülke, J. (2014). SubtiWiki – a database for the model organism
tools to design microbes for the production of chemicals and fuels.
Biotechnol Adv 31, 811–817.
Bacillus subtilis that links pathway, interaction and expression
information. Nucleic Acids Res 42 (D1), D692–D698.
Shi, S., Chen, T., Zhang, Z., Chen, X. & Zhao, X. (2009).
Mizoguchi, H., Mori, H. & Fujio, T. (2007). Escherichia coli minimum
Transcriptome analysis guided metabolic engineering of Bacillus
subtilis for riboflavin production. Metab Eng 11, 243–252.
genome factory. Biotechnol Appl Biochem 46, 157–167.
Simonen, M. & Palva, I. (1993). Protein secretion in Bacillus species.
Mizoguchi,
H.,
Sawano,
Y.,
Kato,
J.
&
Mori,
H.
(2008).
Microbiol Rev 57, 109–137.
Superpositioning of deletions promotes growth of Escherichia coli
with a reduced genome. DNA Res 15, 277–284.
Tanaka, K., Henry, C. S., Zinner, J. F., Jolivet, E., Cohoon, M. P., Xia,
F., Bidnenko, V., Ehrlich, S. D., Stevens, R. L. & Noirot, P. (2013).
Morimoto, T., Kadoya, R., Endo, K., Tohata, M., Sawada, K., Liu, S.,
Ozawa, T., Kodama, T., Kakeshita, H. & other authors (2008).
Building the repertoire of dispensable chromosome regions in Bacillus
subtilis entails major refinement of cognate large-scale metabolic
model. Nucleic Acids Res 41, 687–699.
Enhanced recombinant protein productivity by genome reduction in
Bacillus subtilis. DNA Res 15, 73–81.
Moya, A., Gil, R., Latorre, A., Peretó, J., Pilar Garcillán-Barcia, M. & de
la Cruz, F. (2009). Toward minimal bacterial cells: evolution vs.
Touchon, M., Hoede, C., Tenaillon, O., Barbe, V., Baeriswyl, S., Bidet,
P., Bingen, E., Bonacorsi, S., Bouchier, C. & other authors (2009).
design. FEMS Microbiol Rev 33, 225–235.
Organised genome dynamics in the Escherichia coli species results in
highly diverse adaptive paths. PLoS Genet 5, e1000344.
Nandagopal, N. & Elowitz, M. B. (2011). Synthetic biology: integrated
Trinh, C. T., Unrean, P. & Srienc, F. (2008). Minimal Escherichia coli
gene circuits. Science 333, 1244–1248.
cell for the most efficient production of ethanol from hexoses and
pentoses. Appl Environ Microbiol 74, 3634–3643.
Nicolas, P., Mäder, U., Dervyn, E., Rochat, T., Leduc, A., Pigeonneau,
N., Bidnenko, E., Marchadier, E., Hoebeke, M. & other authors
(2012). Condition-dependent transcriptome reveals high-level reg-
ulatory architecture in Bacillus subtilis. Science 335, 1103–1106.
O’Connor, M. & Gregory, S. T. (2011). Inactivation of the RluD
Wang, Q., Zhang, Y., Yang, C., Xiong, H., Lin, Y., Yao, J., Li, H., Xie, L.,
Zhao, W. & other authors (2010). Acetylation of metabolic enzymes
coordinates carbon source utilization and metabolic flux. Science 327,
1004–1007.
pseudouridine synthase has minimal effects on growth and ribosome
function in wild-type Escherichia coli and Salmonella enterica.
J Bacteriol 193, 154–162.
Westers, H., Dorenbos, R., van Dijl, J. M. & other authors (2003).
Park, S. J., Lee, T. W., Lim, S. C., Kim, T. W., Lee, H., Kim, M. K., Lee,
S. H., Song, B. K. & Lee, S. Y. (2012). Biosynthesis of polyhydrox-
Yano, K., Wada, T., Suzuki, S., Tagami, K., Matsumoto, T., Shiwa, Y.,
Ishige, T., Kawaguchi, Y., Masuda, K. & other authors (2013).
yalkanoates containing 2-hydroxybutyrate from unrelated carbon
source by metabolically engineered Escherichia coli. Appl Microbiol
Biotechnol 93, 273–283.
Multiple rRNA operons are essential for efficient cell growth and
sporulation as well as outgrowth in Bacillus subtilis. Microbiology 159,
2225–2236.
Pitera, D. J., Paddon, C. J., Newman, J. D. & Keasling, J. D. (2007).
Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab Eng 9, 193–207.
Yim, H., Haselbeck, R., Niu, W., Pujol-Baxley, C., Burgard, A., Boldt,
J., Khandurina, J., Trawick, J. D., Osterhout, R. E. & other authors
(2011). Metabolic engineering of Escherichia coli for direct production
Pósfai, G., Plunkett, G., Fehér, T. & other authors (2006). Emergent
of 1,4-butanediol. Nat Chem Biol 7, 445–452.
properties of reduced-genome Escherichia coli. Science 312, 1044–
1046.
Yu, B., Sung, B., Koob, M., Lee, C., Lee, J., Lee, W., Kim, M. & Kim, S.
(2002). Minimization of the Escherichia coli genome using a Tn5-
Genome engineering reveals large dispensable regions in Bacillus
subtilis. Mol Biol Evol 20, 2076–2090.
Quan, J. & Tian, J. (2009). Circular polymerase extension cloning of
targeted Cre/loxP excision system. Nat Biotechnol 20, 1018–1023.
complex gene libraries and pathways. PLoS ONE 4, e6441.
Quan, J. & Tian, J. (2011). Circular polymerase extension cloning
Yu, B. J., Kang, K. H., Lee, J. H., Sung, B. H., Kim, M. S. & Kim, S. C.
(2008). Rapid and efficient construction of markerless deletions in the
for high-throughput cloning of complex and combinatorial DNA
libraries. Nat Protoc 6, 242–251.
Zhang, Y., Werling, U. & Edelmann, W. (2012). SLiCE: a novel
Rasko, D. A., Rosovitz, M. J., Myers, G. S., Mongodin, E. F., Fricke,
W. F., Gajer, P., Crabtree, J., Sebaihia, M., Thomson, N. R. & other
authors (2008). The pangenome structure of Escherichia coli:
comparative genomic analysis of E. coli commensal and pathogenic
isolates. J Bacteriol 190, 6881–6893.
Rasko, D. A., Webster, D. R., Sahl, J. W., Bashir, A., Boisen, N.,
Scheutz, F., Paxinos, E. E., Sebra, R., Chin, C.-S. & other authors
(2011). Origins of the E. coli strain causing an outbreak of hemolytic-
uremic syndrome in Germany. N Engl J Med 365, 709–717.
Sandmann, G. (2002). Combinatorial biosynthesis of carotenoids in a
heterologous host: a powerful approach for the biosynthesis of novel
structures. ChemBioChem 3, 629–635.
Escherichia coli genome. Nucleic Acids Res 36, e84.
bacterial cell extract-based DNA cloning method. Nucleic Acids Res
40, e55.
Zhou, L., Niu, D. D., Tian, K. M., Chen, X. Z., Prior, B. A., Shen, W., Shi,
G. Y., Singh, S. & Wang, Z. X. (2012a). Genetically switched D-lactate
production in Escherichia coli. Metab Eng 14, 560–568.
Zhou, L., Tian, K. M., Niu, D. D., Shen, W., Shi, G. Y., Singh, S. & Wang,
Z. X. (2012b). Improvement of D-lactate productivity in recombinant
Escherichia coli by coupling production with growth. Biotechnol Lett
34, 1123–1130.
Zweers, J. C., Barák, I., Becher, D., Driessen, A. J., Hecker, M.,
Kontinen, V. P., Saller, M. J., Vavrová, L. & van Dijl, J. M. (2008).
Schallmey, M., Singh, A. & Ward, O. P. (2004). Developments in the
Towards the development of Bacillus subtilis as a cell factory for
membrane proteins and protein complexes. Microb Cell Fact 7, 10.
use of Bacillus species for industrial production. Can J Microbiol 50,
1–17.
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Downloaded from www.microbiologyresearch.org by
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