Journal of Bioscience and Bioengineering

VOL. xx No. xx, 1e9, 2015

www.elsevier.com/locate/jbiosc

REVIEW

Exploring the proteomic characteristics of the Escherichia coli B and K-12 strains in
different cellular compartments

Mee-Jung Han*

Department of Biomolecular and Chemical Engineering, Dongyang University, 145 Dongyang-daero, Punggi-eup, Yeongju, Gyeongbuk 750-711, Republic of Korea

Received 17 September 2015; accepted 3 December 2015

Available online xxx
Escherichia coli, one of the well-characterized prokaryotes, has been the most widely used bacterial host in scientific
studies and industrial applications. Many different strains have been developed for the widespread use of E. coli in
biotechnology, and selecting an ideal host to produce a specific protein of interest is a critical step in developing a
production process. The E. coli B and K-12 strains are among the most frequently used bacterial hosts for the production
of recombinant proteins as well as small-molecule metabolites such as amino acids, biofuels, carboxylic acids, diamines,
and others. However, both strains have distinctive differences in genotypic and phenotypic attributes, and their be-
haviors can still be unpredictable at times, especially while expressing a recombinant protein. Therefore, in this review,
an in-depth analysis of the physiological behavior on the proteomic level was performed, wherein the particularly
distinct proteomic differences between the E. coli B and K-12 strains were investigated in the four distinctive cellular
compartments. Interesting differences in the proteins associated with key cellular properties including cell growth,
protein production and quality, cellular tolerance, and motility were observed between the two representative strains.
The resulting enhancement of knowledge regarding host physiology that is summarized herein is expected to contribute
to the acceleration of strain improvements and optimization for biotechnology-related processes.
Ó 2015, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Escherichia coli proteome; Comparative proteome; Physiological difference; Subcellular compartment; Biotechnology]

Escherichia coli has been extensively studied and is the most
widely used bacterial host in scientific research and industrial ap-
plications. Specifically, it has been best characterized in terms of its
molecular genetics and physiology, the availability of a large
number of cloning or expression vectors and various mutant
strains, and the development of large-scale cultivation processes
(1,2). The resulting enhanced knowledge regarding E. coli allows us
to better employ it for the production of recombinant proteins as
well as small-molecule metabolites such as amino acids, biofuels,
carboxylic acids, diamines, and others. Additionally, because E. coli
is extensively used in biotechnology, many different strains have
been developed, and selecting an ideal host to produce a specific
protein of interest is a critical step in developing a production
process.
Among these strains are the most frequently used laboratory
strains, E. coli B and K-12. In the past, E. coli B or its related strains
served as the research model for studies of phage sensitivity, re-
striction systems, mutagenic assays, and bacterial evolution, while
the K-12 strain or its derivatives was used for most genetic and
biochemical studies (3,4). One attractive characteristic is that de-
rivatives of E. coli B, especially BL21, have been widely used for the
overproduction of recombinant proteins, biofuels, and other bio-
molecules (2), because of several favorable features including faster
growth and lower acetate accumulation in minimal media, higher
* Tel.: þ82 54 630 1148; fax: þ82 54 630 1275.
E-mail address: mjhan75@dyu.ac.kr.
expression levels of recombinant proteins, and less degradation of
these proteins during purification compared to the K-12 strains (5).
These findings indicate that distinct E. coli strains exhibit different
physiological behaviors and metabolism at the molecular level,
resulting in contributing phenotypic attributes. Thus, intensive
work has been conducted to understand the differences between
the two strains.
Initially, comparisons between both strains were investigated at
the level of phenotypic differences, such as biomass yield, growth
characteristics and acetate production, or production of recombi-
nant proteins (5,6). However, the cultivation characteristics of a
specific host can only provide limited systems information. Sub-
sequently, several studies were performed that utilized metabolic
pathway analysis to follow carbon flow using flux analysis (7) and
to follow individual gene transcription using northern blot analysis
and cDNA microarrays (8). Systems biological research of the E. coli
B and K-12 strains accelerated after the genome sequences of both
strains became publicly available. Overall, the genome sequences of
E. coli B and K-12 are highly similar, but there are numerous strain-
specific gene disruptions caused by deletions, frameshifts, or in-
sertions of insertion sequence (IS) elements that might affect bac-
terial phenotypes (9). Additionally, comparative proteomic studies
between the two strains were conducted by two-dimensional gel
electrophoresis (2-DE) and mass spectrometry (MS) (10e12). More
systematic analyses of the E. coli B and K-12 strains cultured in
shake flasks were performed for the identification of metabolic and
physiological alterations by omics studies, such as investigation of
the genome, transcriptome, proteome, and phenome (13). The

1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2015.12.005

Please cite this article in press as: Han, M.-J., Exploring the proteomic characteristics of the Escherichia coli B and K-12 strains in different cellular
compartments, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.12.005
2 HAN J. BIOSCI. BIOENG.,

majority of the strain-specific phenotypes could be explained by
the variation data, especially from gene distributions and deletions.
Similarly, Marisch et al. (14) compared E. coli B and K-12 hosts
cultured in batch bioreactors on the transcriptome and proteome
levels, revealing distinct differences for the genes and proteins
involved in transport, iron acquisition, and motility. The differences
and similarities established between the strains will provide a
useful baseline for understanding the biology of E. coli strains and
for strain improvement to create a better protein factory. The
context specific proteome, in particular, constitutes the true func-
tional output of a cell and consequently, proteomic characteristics
present a close relationship with the strain-specific phenotype.
Within the past decade, proteomic methods have become
increasingly accessible to researchers, largely due to the robustness
of the analytical methods as well as the affordability of mass
spectrometers and their relative ease of use (15e17). Therefore, this
review is more focused on the strain-specific features based on the
proteomic differences in both hosts, the E. coli B and K-12 strains,
and excludes proteomic technologies or technical limitations
(15e17) as well as the detailed practical applications of proteome
data in biotechnology (15,18,19), which have been nicely reviewed
by several researchers. Instead, several examples for target dis-
covery and new developments in biotechnology and bioengi-
neering based on E. coli proteomic studies were summarized in
Table 1. In some cases that lack proteome data or when the data
were gathered by combined analysis of omics studies, the infor-
mation has also been enhanced by other omics data, particularly
transcriptome analysis.
DISTINCT PROTEOMIC DIFFERENCES BETWEEN E. COLI B AND K-
12 STRAINS
Numerous small- and larger-scale experiments have continually
contributed to expanding the understanding of the nature of whole
protein networks in E. coli. Most proteomic studies were performed
on whole cell extracts without cellular subfractionation. However,
several proteomic studies have been completed using cellular
subfractionation based on the specific properties of proteins from
the different compartments. This approach has the added benefits
of reducing sample complexity, identifying additional unique pro-
teins, localizing newly discovered proteins to specific compart-
ments, and in some cases, allowing functional validation.
The E. coli cell is composed of three soluble compartments
(cytoplasm, periplasm, and extracellular milieu) and two different
membranes (inner and outer membranes). Native or recombinantly
expressed proteins can be directed to these five different locations
in E. coli. Many researchers have developed various strategies to
direct recombinant proteins to different cellular compartments
(1,2). However, the decision to target recombinant proteins toward
any compartment relies on balancing the advantages and disad-
vantages of each compartment. Furthermore, a variety of efficient
methods for protein sample preparation from the different com-
partments have been continually developed and have minimal
cross-contaminations (20e22). Thus, large, extensive subcellular
proteome maps for E. coli B (22) and K-12 (20), including the
cytoplasmic, periplasmic, extracellular and inner and outer mem-
brane proteomes, were established by employing 2-D gels and MS.
In-depth comparative analyses of individual subcellular proteomes
of the E. coli B and K-12 strains, including periplasmic proteomes
(12), membrane proteomes (11), and extracellular proteomes (10),
have been performed to compare both strains. Recently, a sys-
tematic analysis of the two strains was performed with focus on the
metabolic and physiological differences using omics studies,
including genomics, transcriptomics, proteomics, and phenomics
(13). Another comparative analysis by Marisch et al. (14) focused on
strain characteristics and transcriptional and translational patterns
of industrial E. coli B and K-12 strains in high glucose batch culti-
vations, specifically B strain BL21 and K-12 strains HMS174 and
RV308. Therefore, we have delineated the differences between the
E. coli B and K-12 proteomes according to their cellular compart-
ments (Fig. 1).
Cytoplasm The E. coli cytoplasm is the main place where
energy and reducing power are extracted or generated and the
building blocks of cellular macromolecules are synthesized. These
processes are orchestrated by a highly integrated network of
chemical reactions that are collectively known as metabolism or
intermediary metabolism. Therefore, the analysis of the metabolic
differences in bacterial strains is important for the development of
strains with desired growth and production properties.
Typically, total protein extracts without separating only the
cytosolic proteins are prepared for proteomics studies from the
whole cell with addition of a lysis buffer and in some cases, by the
aid of physical power, such as grinding, high-pressure, or sonicat-
ion. Unfortunately, the complete proteome could not be solubilized
with a typical resolving buffer containing urea and/or thiourea. The
dissolved proteins are largely the soluble proteins in the cell, and in
general, less than 600 proteins are annotated from a 2-D gel of
cytoplasmic proteins or a whole cell extract (20,22). Consequently,
the proteome data acquired provide an incomplete story for deci-
phering and understanding complex cellular behaviors or

TABLE 1. Typical examples of E. coli proteome-based applications in biotechnology and bioengineering.

Subcellular proteome Primary purpose Targets to be engineered Species Reference

to be studied

Cytoplasmic (whole cell) Enhanced protein production CysK E. coli B strain BL21(DE3) 29
GlyA E. coli B strain BL21(DE3) 71
HlyA E. coli K-12 strain W3110 72
IbpA, IbpB E. coli K-12 strain W3110 73
PspA E. coli K-12 strain 59A7 74
Enhanced biopolymer production FbaA, TpiA E. coli K-12 strains, W3110 and XL1-Blue lacI mutants, 75
and E. coli W lacI mutant strain
Enhanced protein solubility ArsC E. coli B strain BL21(DE3) 76
Crr, PotD E. coli B strain BL21(DE3) 77
EDA E. coli B strain BL21(DE3) 78
Mdh E. coli B strain BL21(DE3) 79
RpoS E. coli B strain BL21(DE3) 80
SlyD E. coli B strain BL21(DE3) 81
Tsf E. coli B strain BL21(DE3) 82
Discovery of conditional promoter AldA E. coli K-12 strain W3110 83
Discovery of genetic mutation KdgR, DeoR E. coli K-12 strains, XL1-Blue and DH5a 84
Membrane Development of cell surface display OmpX E. coli K-12 strain XL10-Gold 85
Extracellular Enhanced production of excretory protein OsmY E. coli B strain BL21(DE3) 69

Please cite this article in press as: Han, M.-J., Exploring the proteomic characteristics of the Escherichia coli B and K-12 strains in different cellular
compartments, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.12.005
VOL. xx, 2015
FIG. 1. A summary of the distinctive proteomic features of both E. coli B and K-12. The characteristics of the E. coli B and K-12 strains derived from the proteome data are explained for
each cellular compartment. Proteomic differences between the B and K-12 strains are arbitrarily depicted to facilitate explanation. The direction is given by the arrow, while the
length of the arrow represents its magnitude.
metabolism. Therefore, proteomic studies have frequently been
combined with other biochemical or omics tools. Another alter-
native strategy is to use recent quantitative proteomics methods
based on tandem mass spectrometry (MS/MS) (called the non-gel
based approach), including label-free, metabolic labeling (i.e., sta-
ble isotope labeling by amino acids in cell culture or SILAC), and
isobaric chemical labeling [i.e., isobaric tags for relative and abso-
lute quantification (iTRAQ) or tandem mass tag (TMT)] (23).
Isobaric chemical labeling, in particular, is capable of accurate,
precise, and reproducible quantification and provides deep prote-
ome coverage for quantification, even if there are many samples to
analyze. Several groups have used iTRAQ labeling or SILAC tech-
niques for exploring the E. coli proteome (24,25). However, a
comparative physiological study of the E. coli B and K-12 hosts has
not yet been performed using a quantitative proteomic method.
Therefore, while remarkable metabolic differences in overall host
attributes between E. coli B and K-12 exist, we refer to two meta-
bolic pathways (the acetate assimilation pathway and the biosyn-
thetic and degradation pathways of amino acids) in this section,
based on limited proteomic information and other supported
results.
One of the most notable characteristics is that E. coli B produces
much less acetate than E. coli K-12, even in the presence of excess
glucose (5,6). In addition, E. coli B utilizes glucose more efficiently
than E. coli K-12, achieving much higher biomass yields and growth
rates compared to the K-12 strains (13,14). Thus, many studies have
been performed to determine the reason for this difference. The
availability of E. coli genomic sequences and DNA microarray
technology facilitates analysis of the metabolic variations between
the two strains (8,13,14). Additionally, proteomic evidence has
further supported the formation of metabolic differences. The first
plausible explanation of less acetate accumulation in the E. coli B
strains is the activation of the glyoxylate shunt encoded by the
acetate operon (aceBAK). Moreover, Noronha et al. (7) reported that
a higher flux through the tricarboxylic acid cycle (TCA) and a more
active glyoxylate shunt in the B strain compared to K-12 strain
JM109 were responsible for lower acetate levels. However, the
accumulated evidence showed that the aceBAK expression levels in
Please cite this article in press as: Han, M.-J., Exploring the proteomic characteristics of the Escherichia coli B and K-12 strains in different cellular
compartments, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.12.005
PROTEOMIC COMPARISON OF E. COLI B AND K-12 3
the K-12 strains were higher than BL21 in flask cultures (13), batch
fermentation (14) or fed-batch fermentation (8). This finding was
also concordant with proteomics, which found AceA and AceB
present at substantially elevated levels in K-12 strains MG1655 and
W3110 compared to B strains REL606 and BL21(DE3) (13). However,
the acs gene encoding acetyl-CoA synthetase was detected at
higher levels in the B strain (14). Acetyl-CoA synthetase activates
the cellular ability to convert acetate to acetyl-CoA. Higher acs
expression was induced by high cAMP levels in the B strain (26),
most likely contributing to lower acetate accumulation in the B
strain compared to the K-12 strains. Furthermore, recent results
suggest that overexpression of the non-coding small RNA SgrS
enables E. coli B to reduce its acetate excretion by controlling
glucose transport when cells are exposed to high glucose concen-
trations (27). This is an additional mechanism that allows the E. coli
B strain to respond better to high glucose concentrations and
consequently lower acetate excretion. Thus, lower acetate accu-
mulation during cultivation may enable the B strain to reach higher
growth rates, because acetate inhibits cellular growth and foreign
protein production (5,28).
In general, the levels of enzymes involved in the biosynthesis of
specific amino acids in E. coli are decreased during the production
of recombinant proteins (29) or high cell density cultivation (30).
This response may vary to differing degrees according to the host
strain, the target protein being produced, or the culture conditions.
Thus, it may be difficult to find a general rule or certain tendency in
amino acid biosynthesis, especially during recombinant protein
expression. However, comparative proteome analysis of the E. coli B
and K-12 hosts showed that most of enzymes required for the
biosynthesis of arginine (ArgDCI), serine (SerC), and aspartate
(AspC) were synthesized at high levels in rich media for the B strain
in shaker flasks (13). This was consistent with the transcriptome
results, which indicated that the genes required for arginine and
branched-chain amino acids (leucine, isoleucine, and valine)
biosynthesis were more highly expressed in B than in K-12. In
contrast, the study revealed that TnaA, a protein involved in the
tryptophan and cysteine degradation pathway, was highly
expressed at both mRNA and protein levels in K-12 (13). The same
4 HAN
expression pattern was true for TdcE and TdcF that are required for
anaerobic degradation of L-threonine, GadB that is involved in
glutamate degradation and acid resistance, and AspA that is needed
for the reversible conversion of L-aspartate to fumarate and
ammonia. These observations suggest that B strains are, in general,
evolved for enhanced amino acid biosynthesis and reduced
degradation, which is favorable for the efficient production of re-
combinant proteins.
For the high-level production of specific amino acids, it would
be a beneficial feature for the E. coli to have feedback inhibition-
resistance by genetic mutation (31). It is appropriate for the first
enzyme or the enzyme at pathway branch points in the biosyn-
thetic pathways of amino acids to be allosterically controlled by
feedback inhibition and transcriptional attenuation. By analysis of
the genomic sequences of the E. coli strains, it would be easy to find
mutations in these specific genes (32). For example, wild-type
E. coli K-12 contains a frameshift site within a gene (ilvG) that is
intact in B, and therefore the K-12 strain mediates valine resistance,
permitting growth on various valine dipeptides (9,13,33). Thus,
almost all proteinogenic amino acids, with a few exceptions, can be
produced industrially by specially developed mutants of E. coli with
a feedback inhibition-resistance or mutation in the exporter and
transporter systems by random mutagenesis and rational meta-
bolic engineering (31), rather than the use of a host with specific
physiological or metabolic characteristics.
Periplasm The estimated width of the E. coli periplasm is
26  5 nm, which is equivalent to 10e13% of its total cell volume
(34). The narrow periplasmic space is rich in proteins and
carbohydrates. Specifically, it contains enzymes that catalyze the
formation and rearrangement of disulfide bonds.
There are two typical isolation methods of periplasmic proteins,
which include the cold osmotic shock method (35) or cell spher-
oplasting followed by differential centrifugation (36). Lopez-
Campistrous et al. (20) identified 60 different periplasmic pro-
teins from E. coli K-12. The top 5 major proteins of the E. coli K-12
strains are OppA, DppA, GlnH, MalE, and FliY, which are periplas-
mic substrate-binding proteins for transport across cell mem-
branes. Additionally, Han et al. (22) annotated 45 spots and
identified 30 unique periplasmic proteins from the E. coli B strain.
However, a few of the cytosolic proteins were contaminants in the
periplasm fraction. Recently, Han et al. (12) improved the isolation
method of periplasmic proteins using cold osmotic shock. Using
this method, the largest number of periplasmic proteomes of the
E. coli B and K-12 strains were well-defined with minimal cytosolic
contamination, resulting in the identification of the localization of
many new proteins, including 30 proteins from the K-12 strain and
53 proteins from the B strain, to periplasmic origin by comparison
with two previous datasets (20,22). The highly abundant proteins
were HdeA, HdeB, MalE, MglB, and OppA for K-12 MG1655, while it
was Agp, FkpA, GlnH, GltI, MalE, and OppA for BL21(DE3).
Corroborating the results obtained by Lopez-Campistrous et al.
(20), periplasmic binding proteins MalE and OppA were the most
abundant proteins in the two E. coli strains. For comparison, a total
of 50 different proteins were identified as strain-specific proteins
or whose abundance changed significantly between the E. coli K-12
and B strains. Obvious proteomic differences between the two
strains are most likely caused by the genes, cybC, kpsD, and ybl119
for the K-12 strain and fliC, fliY, mglB, and modA for the B strain, that
are missing. The major functional differences in the E. coli peri-
plasmic components were categorized into three groups: enzymes,
transporters, and uncharacterized proteins (12). Several hydro-
lases, including hydrolytic enzymes, glycosidases, folding catalytic
enzymes, and proteases, were synthesized more in the B strain
than in the K-12 strain. Additionally, transporters, which typically
belong to the ATP-binding cassette (ABC) transport systems, are
Please cite this article in press as: Han, M.-J., Exploring the proteomic characteristics of the Escherichia coli B and K-12 strains in different cellular
compartments, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.12.005
J. BIOSCI. BIOENG.,
specifically synthesized in each strain in a complementary manner
(see below). Intriguingly, a significant number of proteins in the
periplasm of E. coli have no annotated function. Thus, we still need
to study the functions that these proteins have in the periplasmic
space.
An intriguing property of the E. coli periplasm is that numerous
chaperones, folding catalysts and proteases exist and cooperate in
protein folding and protein quality control in this cellular
compartment. Therefore, in contrast to cytosolic production, peri-
plasmic secretion of heterologous proteins offers several advan-
tages, which include improved folding, enhanced solubility,
prevention of protease degradation, N-terminal authenticity, and
ease of purification (2). Han et al. (12) reported a comparative
analysis of the E. coli B and K-12 strains, which indicated higher
expression levels of thiol-disulfide oxidoreductase DsbC and two
peptidyl-prolyl cisetrans isomerases (PPIases), FkpA and PpiA, in
the B strain compared to the K-12 strain. Additionally, two pepti-
dases, Prc and PtrA, were highly expressed in the E. coli B strain.
Evidence suggests that the aforementioned proteins help enhance
the secretion of functional proteins in E. coli. In some cases, coex-
pression with these periplasmic chaperones may enhance the
production of recombinant proteins in E. coli. Reilly and Yansura
(37) reported that overexpression of DsbA and DsbC improved the
efficiency of the assembly of the light and heavy chains of a full-
length antibody in the periplasm. Another study reported that
coexpression of periplasmic chaperones Skp and/or FkpA increased
the solubility of antibody fragments and cell viability in E. coli
BL21(DE3) (38). Moreover, in BL21, insertion elements have func-
tionally disabled two proteases, the major cytoplasmic protease Lon
(encoded by lon) and the outer membrane protease OmpT (encoded
by ompT) (32). In fact, the Lon protease deficiency in the B strains is
one reason that they have been developed as E. coli hosts of choice
for the overproduction of recombinant proteins.
Furthermore, two acid stress periplasmic chaperones, HdeA and
HdeB (39), which prevent periplasmic protein aggregation at acidic
pH values were not detected in the B strain but were highly syn-
thesized in the K-12 strain only (12). Accordingly, the expression
levels of cytoplasmic chaperones DnaK and ClpB, which correlate
with cellular stress levels, are higher in the K-12 strains (JM105,
HB101, and TOP10) than in the BL21 strains (40), indicating that
E. coli B may be more susceptible than K-12 to certain stress con-
ditions, which is consistent with the higher membrane perme-
ability discussed later. Taken together, this evidence suggests that
E. coli B has an advantage in protein secretion, while the K-12 strain
possesses a greater tolerance.
Another property of the E. coli periplasm is periplasmic
substrate-binding proteins, which are the initial receptors in the
process of active transport across cell membranes and/or chemo-
taxis. These proteins bind a variety of ligands, such as sugars, amino
acids, peptides, ions, and vitamins, with high affinity (41). An
interesting detail about the two E. coli strains is that the maltose-
binding protein (MBP) MalE and the oligopeptide-binding protein
OppA are the most abundant periplasmic proteins. Specifically,
E. coli can make MalE to be used as a fusion partner for the secretion
of recombinant proteins to increase the solubility of intracellular
proteins and to improve the export of secreted proteins in bacterial
systems (42,43). MBP is clearly a spectacular solubilizing agent, but
there is also evidence to suggest that it may be able to function as a
general molecular chaperone in the context of a fusion protein that
can temporarily sequester aggregation-prone folding intermediates
of its fusion partners and prevent their self-association. Richarme
and Caldas (44) showed that OppA and MalE from E. coli and
galactose-binding protein MglB from Salmonella typhimurium
interact with unfolded and denatured proteins much like the mo-
lecular chaperones that are involved in protein folding and protein
renaturation after stress.
VOL. xx, 2015
Interestingly, it was reported that many phenotypic differences
between the K-12 and B strains in the utilization of carbon sources
might be correlated with genotypic differences (13). ModA, which
is the periplasmic binding component of the high-affinity molyb-
date ABC transporter, is only detected in K-12 MG1655. This is
consistent with the presence of the modABC operon in the K-12
strain but not the B strain. Specifically, MglB and YtfQ are the
periplasmic binding components of galactose ABC transporters
MglABC and YtfQRT/YjfF, respectively. MglB is synthesized only in
the K-12 strain (12), as the gene does not exist in the B strain. This
protein has been correlated with the phenotypic behavior that the
K-12 strain could more readily use galactose than the B strain (13).
However, the uncharacterized YtfQ protein was only detected in the
B strain (12). This opposite expression of MglB and YtfQ in the two
strains revealed that one of the transport systems may be domi-
nantly and specifically regulated for efficient uptake of galactose
components in a strain-specific manner. Induction of the galactose
ABC transport system has been observed in glucose-limited cul-
tures to optimize carbon transport (45). Additionally, MalM, which
is encoded by the last gene in the malK-lamB-malM operon, un-
derwent increased synthesis in B strain compared to the K-12
strain. Thus, the maltose system is adapted to scavenge for maltose
and maltodextrins, particularly when fasting on glucose or other
carbohydrate carbon sources (46). These findings are consistent
with the results of transcriptome analysis, where the gene
expression levels for maltose ABC transporter malKFGE and galac-
tose ABC transporter mglABC were higher in the BL21 strain (14).
Presumably, different substrate transport mechanisms and higher
cAMP levels enabled higher growth rates of the BL21 strain.
Furthermore, GlnH, the periplasmic glutamine-binding protein of
the GlnHPQ high-affinity glutamine ABC transporter, and GltI, the
periplasmic glutamate-binding component of the GltIJKL glutamate
ABC transporter, are synthesized at high levels in the BL21(DE3)
strain compared to the K-12 strain (12). The enhanced trans-
portation and synthetic amino acid pathways as well as the lower
accumulation of acetate in the B strains (as described above) pro-
vide other beneficial features for cell growth and protein
production.
Membrane Approximately 30e40% of all E. coli proteins may
function in the membrane of the cell envelope (47). They have
important roles in essential cellular processes, including cell wall
assembly, the synthesis and remodeling of peptidoglycan,
nutrient uptake, energy production, adherence, motility,
environmental sensing, virulence, and antibiotic resistance.
Several studies have shown that the synthesis of outer membrane
proteins (OMPs) changes when bacteria are exposed to
environmental changes from various stresses (48). In particular,
the outer membrane contributes to resistance, as the narrow
porin channels slow down the penetration of even small,
hydrophilic solutes and the low fluidity of the lipopolysaccharide
(LPS) leaflet decreases the transmembrane diffusion rate of
lipophilic solutes.
Because of the technical aspects involved, the membrane pro-
teome has been a challenge to study until very recently. Multiple
approaches have been newly developed to efficiently isolate the
membrane proteins with minimal contamination. One of these
methods has been widely used for 2-D gel based proteome analysis
and allows the E. coli membrane proteins to be readily isolated by
sodium carbonate or sodium lauroyl sarcosinate (sarcosine)
enrichment (18). Furthermore, the inner membrane proteins (IMPs)
and OMPs can then be separated by sucrose density gradient
centrifugation. Huang et al. (49) identified 31 OMPs enriched in the
sarcosine-insoluble fraction that included previously unrecognized
membrane-interacting protein complexes, such as the complex
consisting of OmpW and fumarate reductase. Numerous IMPs and
Please cite this article in press as: Han, M.-J., Exploring the proteomic characteristics of the Escherichia coli B and K-12 strains in different cellular
compartments, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.12.005
PROTEOMIC COMPARISON OF E. COLI B AND K-12 5
OMPs were also identified from E. coli K-12 (20) and B REL606
strains (22) by using sucrose density gradient centrifugation.
Recent high-throughput studies have focused in particular on
the E. coli complexome, which is defined as the set of protein
complexes. In fact, the entire cell can be viewed as a factory that
contains an elaborate network of interlocking assembly lines, each
of which is composed of a set of large protein machines. This is
particularly true in cellular membranes, where many well-
characterized proteins assemble into complexes that carry out
important tasks in energy generation, protein trafficking, and small
molecule transport. Stenberg et al. (47) identified 43 distinct pro-
tein complexes, including 34 distinct IMPs and 9 different OMPs, in
the cell envelope of the E. coli B strain using blue native-
polyacrylamide gel electrophoresis (BN-PAGE) in combination
with SDS-PAGE and MS. Additionally, Maddalo et al. (50) isolated
native membrane protein complexes by a three-step fractionation
with sucrose gradient centrifugation, and then anion exchange
chromatography, followed by BN-PAGE analysis. This research
demonstrated that six (YhcB, YgiM, YggE, YfhM, SlyB, and YfgM) of
the 30 cell envelope complexes identified by MS were newly
identified. Studies such as these facilitate the understanding of the
assembly and composition of E. coli membrane protein complexes
during different growth conditions and in different mutant
backgrounds.
Recent comparative analysis of E. coli membrane proteomes
showed that the B and K-12 strains had somewhat different types
and expression profiles of the protein components in the cell
membrane (11). Of the 165 protein spots, including 62 non-
redundant annotated proteins, four proteins (FliC, Flu, OmpT, and
OmpW) are definitely K-12 strain-specific membrane proteins.
These findings are consistent with genome sequence data that the
corresponding genes are missing in the B strain genome (9).
Furthermore, this research reveals that the E. coli membrane
components predominantly consist of b-barrel proteins, such as
porins, and lipid-modified proteins, the so-called lipoproteins, are
differentially synthesized between the E. coli B and K-12 strains
(11). In particular, the components of the b-barrel proteins in the
E. coli membrane, such as FadL, LamB, OmpA, OmpC, OmpX and
OmpW, have been widely used as anchoring motifs for the display
of recombinant proteins on the E. coli cell surface.
One of distinct characteristics in E. coli membrane is cell
motility. The bacterial flagellum, which is responsible for motility
and behavior and is one of the most sophisticated self-assembling
molecular machines, is embedded in the bacterial cell envelope.
Using the bacterial flagellar motor, which is powered by the proton-
or the sodium-motive force, cells swim in liquids or swarm on
surfaces to move toward favorable environments. Extensive genetic
and biochemical studies of the flagellum have been conducted in
S. typhimurium and E. coli, and more than 50 gene products are
known to be involved in flagellar assembly and function (51).
Furthermore, a sophisticated chemotaxis signaling system allows
the cell to sense chemical stimuli and transmit this information
through a signal transduction cascade that regulates the direction
of flagellar rotation.
The diverse chemotaxis and motility behaviors for the K-12
strains were demonstrated at the proteome level. Several proteo-
mic studies showed that proteins involved in motility and
chemotaxis were highly expressed in E. coli K-12 strains but mostly
had no detectable expression level in B strains (11,13,14). Han et al.
(11) showed that motility- (FliC) and chemotaxis-related proteins
(CheA and CheW) were detected only in K-12 flask cultures in
complex LB medium. Marisch et al. (14) also reported that YcgR,
FliD, and FliY, which are involved in flagellar motility and ma-
chinery, were found in the proteome of only E. coli K-12 HMS174
grown in batch cultivation with semi-complex media. Interestingly,
another study (13) showed that the high quantities of motility-
6 HAN
related proteins (FimA, FlgF, FlgL, FliC, and FliD) were only detected
in the K-12 strains grown in defined media, but they were mostly
secreted into the culture medium. The resulting data revealed that
the detection of different motility-related proteins is dependent
upon culture conditions, but they can only be synthesized in the
E. coli K-12 strains. These findings also corroborate the genomic
information that E. coli B strains lack the flagellar biosynthesis gene
cluster, because a 38-kb region in K-12 from yecF to yedS containing
the fliYZACDSTEFGHIJKLMNOPQR genes is deleted from the B
genome (9), possibly indicating that E. coli B is non-motile. More-
over, E. coli B did not swarm in a swarming motility assay (52). This
further illustrates that the genotype of E. coli is correlated with its
phenotypic properties. Additionally, flagellar biosynthetic genes
are differentially expressed at low pH values (53), where low pH
contributes to the proton-motive force that drives flagellar rotation
(54). This is reflected in the surprisingly different compositions and
protein interaction patterns of flagella from different species, which
may reflect adaptations to species-specific motility needs.
Moreover, the chemotaxis-related proteins (CheA, CheW, and
CheY) are synthesized at a significant level by the K-12 strain
(11,13). This corroborates microarray data for the expression levels
of the chemotaxis genes (cheZYRWA, tap, trg, and tsr) and flhC
(encodes the master regulator of flagellar biosynthesis), which are
much higher in K-12 than in B (13). Thus, the lack of flagellar
biosynthetic genes and the low transcriptional and translational
expression of motility-related genes in the E. coli B strain (13) are
important properties to consider when the B strain is used as a cell
factory in controlled culture conditions, because flagellar biosyn-
thesis is energy-intensive and not necessary under the industrial
setup of constant agitation and a generous supply of nutrients (55).
Furthermore, the reduced genome strain achieved by eliminating
potentially nonessential genes, such as many genes related to the
synthesis of flagella and fimbriae, in E. coli K-12 MG1655 has proven
beneficial for protein production (56). Conversely, it shows that the
K-12 strain with the components of the flagella may be more
adaptable to a natural environment with constantly changing
nutrient conditions and/or stresses. These results are correlated
with the finding that the K-12 strain possesses a greater tolerance
than the B strain, as discussed below.
Typically, the B strains have been widely used for mutagenic
assays and toxicological studies, because they show higher mem-
brane permeability than the K-12 strains (57). The reasons for this
choice can be explained by the following proteomic observation:
E. coli B produces significantly more OmpF porins with a larger pore
size than K-12 (11,13), because the B genome lacks micF, which
post-transcriptionally prevents the production of OmpF (58),
indicating an increase in membrane permeability. Additionally,
most of the lipoproteins (particularly the Bam complex), which
facilitate the assembly of the outer membrane b-barrel proteins, are
strongly induced in B compared to K-12.
In contrast, OmpT and OmpW are synthesized only in E. coli K-12
MG1655 and are not observed in E. coli BL21(DE3), as the corre-
sponding genes are absent from the E. coli B strain genome (11).
This suggests that OmpW is involved in bacterial adaptation in
response to various stresses (59). These results are consistent with
phenotype microarray results indicating that the B strain was more
susceptible than the K-12 strain to a variety of stressful conditions
caused by osmolarity, pH, or exposure to inhibitory compounds
such as salicylate and b-lactam antibiotics (13). Additionally, OmpA
was present in E. coli K12 at higher levels than in E. coli B. The
proposed function of OmpA is as a contributor to the structural
integrity of the outer membrane as well as murein lipoprotein and
peptidoglycan-associated lipoprotein. These strain-specific differ-
ences may influence the permeability and integrity of the cell
membrane, revealing why the E. coli B strain has a higher mem-
brane permeability than E. coli K-12, while the K-12 strain possesses
Please cite this article in press as: Han, M.-J., Exploring the proteomic characteristics of the Escherichia coli B and K-12 strains in different cellular
compartments, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.12.005
J. BIOSCI. BIOENG.,
greater tolerance than the B strain due to reduced membrane
permeability and enhanced resistance. However, AcrA and TolC,
components of a major multidrug efflux transport complex, are
increased in B compared to K-12. This means that the E. coli B strain
may positively control the expression of the AcrAB efflux pump to
offset the lack of resistance relative to K-12 (11).
Extracellular space It is generally assumed that laboratory
strains are unable to secrete many proteins, because the gsp operon,
encoding putative type II secretion machinery, is silenced under
standard laboratory conditions by nucleoid-structuring protein H-
NS (60). Only a small number of proteins, such as hemolysin, are
released into the extracellular space (61). However, recent
proteomic studies show that the laboratory strains of E. coli can
excrete trace amounts of many proteins beyond the inner and
outer membranes under normal culture conditions (10,62),
compared to the natural capacity of Gram-positive bacteria, i.e.,
Bacillus subtilis, to highly secrete various proteins into the
extracellular space. Moreover, E. coli strains secrete greater
quantities of proteins when grown in minimal media, and many
of these proteins have been reported as growth phase-dependent
(10), where they are significantly elevated during stationary
phase (10,13) and strictly regulated by environmental signals or
under quorum-sensing control. Interestingly, the E. coli B strains
are capable of releasing greater amounts of proteins than the K-
12 strains (10,13). Genomic information revealed that the B
genome has an additional gene cluster (gspDEFG) for type II
secretion (T2S) that consists of the homologs of
gspOCDEFGHIJKLM, which is not present in K-12 (9). The existence
of a second T2S secretion system and thus enhanced capability
for protein release suggest that B might be better suited for the
extracellular production of recombinant proteins.
Proteome-level identification of E. coli excreted proteins was
first reported by Nandakumar et al. (62), and they identified that 44
proteins were released from E. coli K-12 during shake flask culti-
vation. Later, it was found that a total of 83 unique proteins were
released from high-cell density cultivation of both the E. coli B and
K-12 strains (10). OmpF is released in large amounts into the culture
medium during high cell density cultivation of E. coli BL21 strains
(10,63). Another distinct difference is the motility-related proteins
(FimA, FlgF, FlgL, FliC, and FliD), which were secreted by only K-12
in flask cultures with chemically defined medium (13), as
mentioned in the cell membrane protein section. Additionally,
FimA and FimF, components of the fimbrial complex, were released
only from K-12 during batch cultivation in chemically defined
medium (10). Antigen 43 (Flu), which may function as a cellular
adhesion component or in the control of colony form variation and
autoaggregation, can also be released from K-12 or by mild heat
treatment (64). This component seems to be one of the main rea-
sons for the different permeability and integrity of cell membranes
in the different E. coli strains, and consequently for the release of
different proteins into the culture medium. All proteome studies of
E. coli strains show that the majority of proteins excreted under
laboratory culture conditions contain secretory proteins, such as
periplasmic and outer membrane proteins (10,62). This means that
the release mechanism in E. coli is not mediated by the typical
secretion systems, such as the Sec-dependent pathway or the twin-
arginine translocation (Tat) pathway. Rather, these proteins
mediate a fundamentally distinct excretion process, the so-called
‘vesicle release’, in E. coli.
Vesicle release under normal growth conditions by both path-
ogenic and non-pathogenic gram-negative bacteria is a ubiquitous
process (65). These nanosized membrane vesicles are spherical,
bilayered proteolipids that harbor specific subsets of proteins,
DNAs, RNAs, and lipids. The vesicle is mainly composed of outer
membrane and periplasmic materials, which are released from the
VOL. xx, 2015
bacterial surface without a loss in membrane integrity (65). Recent
research has facilitated conceptual advancements in this emerging
field, which indicate that extracellular vesicles act as intercellular
communicasomes by transferring signals to their target cell via
surface ligands and delivering receptors and functional molecules
(66). These observations suggest that the vesiculation process can
act to selectively eliminate unwanted material to alleviate envelope
stress, where the material that stresses the envelope can be pack-
aged as cargo into these vesicles and exported out of the cell.
In biotechnology, the importance of these excretory proteins
has been recognized in a variety of E. coli strains, because these
proteins could potentially be used as excretion fusion partners for
the extracellular production of recombinant proteins (18). OmpF, a
highly abundant extracellular protein in E. coli BL21(DE3), was
successfully used for the secretion of recombinant human b-
endorphin into culture medium (63). Similarly, the excreted pro-
tein YebF, which was identified in the supernatant of an E. coli
HB101 culture, was used as a fusion partner for the extracellular
production of recombinant proteins (67). Another study demon-
strated that recombinant proteins fused to the 50 untranslated re-
gion of FliC were secreted into the medium via a modified flagellar
type III secretion apparatus (68). The largest number of potential
fusion partners for the excretory production of recombinant pro-
teins was investigated by Qian et al. (69) from the extracellular
proteome of E. coli BL21(DE3). Among them, the most efficient
excreting fusion partner, OsmY, has been used to excrete heterol-
ogous proteins, such as E. coli alkaline phosphatase, B. subtilis a-
amylase, and human leptin, into the growth media. Therefore,
extracellular proteomic data are not only very important to un-
derstanding the phenotypic characteristics of the cell but can also
be used to develop a system for the high-level extracellular pro-
duction of recombinant proteins from bacteria. Most importantly,
the E. coli B strain has enhanced capacity for protein release into the
extracellular space as well as protein secretion into the periplasmic
space.
CONCLUSIONS AND FURTHER PERSPECTIVE
Although the E. coli B and K-12 strains were derived from the
same Escherichia ancestor (70), it is evident by genome analysis that
they have quite different genotypic properties. By the process of
evolution, these E. coli species have accumulated different net-
works to compensate for their weaknesses, resulting in their
distinct genotypes and/or phenotypes. Based on proteomics data,
the distinctive differences between the E. coli B and K-12 strains
(Fig. 1) are that E. coli B has a greater capacity for acetate assimi-
lation and amino acid biosynthesis and that it lacks proteases and
flagella, making it suitable for the enhanced production of recom-
binant proteins, as it does not consume the extra energy required
by the bacterial flagellar motor. Moreover, the B strain has more
membrane permeability and an additional secretion system, mak-
ing it more favorable for protein secretion. In contrast, the K-12
strain has a rigid membrane structure with flagellar motility and
higher levels of stress-responsive proteins, showing resistance
under stress conditions.
However, proteomics alone does not allow a holistic view of the
overall state of the cell, considering the enormous structural and
functional diversity of proteins. Therefore, the integration of mul-
tiple omics results is important. This type of integrated analysis will
lead to an improved understanding of cellular physiology and
metabolism at the systems level and will pave the way towards
more efficient metabolic engineering. However, how the differing
and nonlinearly correlated omics data can be combined in the real
world has barely been addressed. Nonetheless, this comparative
and integrative systems approach provides a high-resolution
Please cite this article in press as: Han, M.-J., Exploring the proteomic characteristics of the Escherichia coli B and K-12 strains in different cellular
compartments, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.12.005
PROTEOMIC COMPARISON OF E. COLI B AND K-12 7
system-wide view and reveals significant insights into why the two
groups manifest numerous distinct phenotypes.
ACKNOWLEDGMENTS
This work was supported by a grant from Dong Yang University
in 2014. This work was also supported by the Basic Science
Research Program (2010-0008826) through the National Research
Foundation of Korea funded by the Ministry of Education, Science
and Technology.
References
1. Makrides, S. C.: Strategies for achieving high-level expression of genes in
Escherichia coli, Microbiol. Rev., 60, 512e538 (1996).
2. Choi, J. H., Keum, K. C., and Lee, S. Y.: Production of recombinant proteins by
high cell density culture of Escherichia coli, Chem. Eng. Sci., 61, 876e885 (2006).
3. Swartz, J. R.: Escherichia coli recombinant DNA technology, pp. 1693e1712, in:
Neidhardt, F. C., Curtiss, I. R., II, Ingraham, J. L., Lin, E. C. C., Low, K. B.,
Magasanik, B., Reznikoff, W. S., Riley, M., Shaechter, M., and Umbarger, H. E.
(Eds.), Escherichia coli and Salmonella typhimurium: cellular and molecular
biology, 2nd ed. ASM Press, Washington, DC (1996).
4. Yoon, S. H., Jeong, H., Kwon, S. K., and Kim, J. F.: Genomics, biological features,
and biotechnological applications of Escherichia coli B: “Is B for better?!”, pp.
1e17, in: Lee, S. Y. (Ed.), Systems biology and biotechnology of Escherichia coli.
Springer, Netherlands (2009).
5. Luli, G. W. and Strohl, W. R.: Comparison of growth, acetate production, and
acetate inhibition of Escherichia coli strains in batch and fed-batch fermenta-
tions, Appl. Environ. Microbiol., 56, 1004e1011 (1990).
6. Shiloach, J., Kaufman, J., Guillard, A. S., and Fass, R.: Effect of glucose supply
strategy on acetate accumulation, growth, and recombinant protein production
by Escherichia coli BL21 (lambdaDE3) and Escherichia coli JM109, Biotechnol.
Bioeng., 49, 421e428 (1996).
7. Noronha, S. B., Yeh, H. J., Spande, T. F., and Shiloach, J.: Investigation of the
TCA cycle and the glyoxylate shunt in Escherichia coli BL21 and JM109 using
(13)C-NMR/MS, Biotechnol. Bioeng., 68, 316e327 (2000).
8. Phue, J. N., Noronha, S. B., Hattacharyya, R., Wolfe, A. J., and Shiloach, J.:
Glucose metabolism at high density growth of E. coli B and E. coli K: differences
in metabolic pathways are responsible for efficient glucose utilization in E. coli
B as determined by microarrays and Northern blot analyses, Biotechnol. Bio-
eng., 90, 805e820 (2005).
9. Jeong, H., Barbe, V., Lee, C. H., Vallenet, D., Yu, D. S., Choi, S. H., Couloux, A.,
Lee, S. W., Yoon, S. H., Cattolico, L., and other 9 authors: Genome sequences
of Escherichia coli B strains REL606 and BL21(DE3), J. Mol. Biol., 394, 644e652
(2009).
10. Xia, X. X., Han, M. J., Lee, S. Y., and Yoo, J. S.: Comparison of the extracellular
proteomes of Escherichia coli B and K-12 strains during high cell density
cultivation, Proteomics, 8, 2089e2103 (2008).
11. Han, M. J., Lee, S. Y., and Hong, S. H.: Comparative analysis of envelope pro-
teomes in Escherichia coli B and K-12 strains, J. Microbiol. Biotechnol., 22,
470e478 (2012).
12. Han, M. J., Kim, J. Y., and Kim, J. A.: Comparison of the large-scale periplasmic
proteomes of the Escherichia coli K-12 and B strains, J. Biosci. Bioeng., 117,
437e442 (2014).
13. Yoon, S. H., Han, M. J., Jeong, H., Lee, C. H., Xia, X. X., Lee, D. H., Shim, J. H.,
Lee, S. Y., Oh, T. K., and Kim, J. F.: Comparative multi-omics systems analysis of
Escherichia coli strains B and K-12, Genome Biol., 13, R37 (2012).
14. Marisch, K., Bayer, K., Scharl, T., Mairhofer, J., Krempl, P. M., Hummel, K.,
Razzazi-Fazeli, E., and Striedner, G.: A comparative analysis of industrial
Escherichia coli K-12 and B strains in high-glucose batch cultivations on pro-
cess-, transcriptome- and proteome level, PLoS One, 8, e70516 (2013).
15. Han, M. J. and Lee, S. Y.: The Escherichia coli proteome: past, present, and
future prospects, Microbiol. Mol. Biol. Rev., 70, 362e439 (2006).
16. Brewis, I. A. and Brennan, P.: Proteomics technologies for the global identi-
fication and quantification of proteins, Adv. Protein Chem. Struct. Biol., 80,
1e44 (2010).
17. Wöhlbrand, L., Trautwein, K., and Rabus, R.: Proteomic tools for environ-
mental microbiology-a roadmap from sample preparation to protein identifi-
cation and quantification, Proteomics, 13, 2700e2730 (2013).
18. Han, M. J., Lee, S. Y., Koh, S. T., Noh, S. G., and Han, W. H.: Biotechnological
applications of microbial proteomes, J. Biotechnol., 145, 341e349 (2010).
19. Han, M. J., Lee, J. W., and Lee, S. Y.: Understanding and engineering of mi-
crobial cells based on proteomics and its conjunction with other omics studies,
Proteomics, 11, 721e743 (2011).
20. Lopez-Campistrous, A., Semchuk, P., Burke, L., Palmer-Stone, T., Brokx, S. J.,
Broderick, G., Bottorff, D., Bolch, S., Weiner, J. H., and Ellison, M. J.:
8 HAN
Localization, annotation, and comparison of the Escherichia coli K-12 proteome
under two states of growth, Mol. Cell Proteomics, 4, 1205e1209 (2005).
21. Thein, M., Sauer, G., Paramasivam, N., Grin, I., and Linke, D.: Efficient sub-
fractionation of gram-negative bacteria for proteomics studies, J. Proteome
Res., 9, 6135e6147 (2010).
22. Han, M. J., Yun, H., Lee, J. W., Lee, Y. H., Lee, S. Y., Yoo, J. S., Kim, J. Y., Kim, J. F.,
and Hur, C. G.: Genome-wide identification of the subcellular localization of
the Escherichia coli B proteome using experimental and computational
methods, Proteomics, 11, 1213e1227 (2011).
23. Li, Z., Adams, R. M., Chourey, K., Hurst, G. B., Hettich, R. L., and Pan, C.:
Systematic comparison of label-free, metabolic labeling, and isobaric chemical
labeling for quantitative proteomics on LTQ Orbitrap Velos, J. Proteome Res.,
11, 1582e1590 (2012).
24. Ping, L., Zhang, H., Zhai, L., Dammer, E. B., Duong, D. M., Li, N., Yan, Z., Wu, J.,
and Xu, P.: Quantitative proteomics reveals significant changes in cell shape
and an energy shift after IPTG induction via an optimized SILAC approach for
Escherichia coli, J. Proteome Res., 12, 5978e5988 (2013).
25. Wu, Q., Yang, A., Zou, W., Duan, Z., Liu, J., Chen, J., and Liu, L.: Transcriptional
engineering of Escherichia coli K4 for fructosylated chondroitin production,
Biotechnol. Prog., 29, 1140e1149 (2013).
26. Kumari, S., Beatty, C. M., Browning, D. F., Busby, S. J., Simel, E. J., Hovel-
Miner, G., and Wolfe, A. J.: Regulation of acetyl coenzyme A synthetase in
Escherichia coli, J. Bacteriol., 182, 4173e4179 (2000).
27. Negrete, A., Majdalani, N., Phue, J. N., and Shiloach, J.: Reducing acetate
excretion from E. coli K-12 by over-expressing the small RNA SgrS,
N. Biotechnol., 30, 269e273 (2013).
28. Eiteman, M. A. and Altman, E.: Overcoming acetate in Escherichia coli re-
combinant protein fermentations, Trends Biotechnol., 24, 530e536 (2006).
29. Han, M. J., Jeong, K. J., Yoo, J. S., and Lee, S. Y.: Engineering Escherichia coli for
increased productivity of serine-rich proteins based on proteome profiling,
Appl. Environ. Microbiol., 69, 5772e5781 (2003).
30. Yoon, S. H., Han, M. J., Lee, S. Y., Jeong, K. J., and Yoo, J. S.: Combined tran-
scriptome and proteome analysis of Escherichia coli during high cell density
culture, Biotechnol. Bioeng., 81, 753e767 (2003).
31. Park, J. H. and Lee, S. Y.: Metabolic pathways and fermentative production of
L-aspartate family amino acids, Biotechnol. J., 5, 560e577 (2010).
32. Studier, F. W., Daegelen, P., Lenski, R. E., Maslov, S., and Kim, J. F.: Under-
standing the differences between genome sequences of Escherichia coli B
strains REL606 and BL21(DE3) and comparison of the E. coli B and K-12 ge-
nomes, J. Mol. Biol., 394, 653e680 (2009).
33. Lawther, R. P., Calhoun, D. H., Adams, C. W., Hauser, C. A., Gray, J., and
Hatfield, G. W.: Molecular basis of valine resistance in Escherichia coli K-12,
Proc. Natl. Acad. Sci. USA, 78, 922e925 (1981).
34. Matias, V. R., Al-Amoudi, A., Dubochet, J., and Beveridge, T. J.: Cryo-trans-
mission electron microscopy of frozen-hydrated sections of Escherichia coli and
Pseudomonas aeruginosa, J. Bacteriol., 185, 6112e6118 (2003).
35. Neu, H. C. and Heppel, L. A.: The release of enzymes from Escherichia coli by
osmotic shock and during the formation of spheroplasts, J. Biol. Chem., 240,
3685e3692 (1965).
36. Köster, W. and Braun, V.: Iron-hydroxamate transport into Escherichia coli
K12: localization of FhuD in the periplasm and of FhuB in the cytoplasmic
membrane, Mol. Gen. Genet., 217, 233e239 (1989).
37. Reilly, D. E. and Yansura, D. G.: Production of monoclonal antibodies in E. coli,
pp. 295e308, in: Shire, S. J., Gombotz, W., Bechtold-Peters, K. and Andya, J.
(Eds.), Current trends in monoclonal antibodies development and
manufacturing. Springer, New York (2010).
38. Ow, D. S., Lim, D. Y., Nissom, P. M., Camattari, A., and Wong, V. V.: Co-
expression of Skp and FkpA chaperones improves cell viability and alters the
global expression of stress response genes during scFvD1.3 production, Microb.
Cell Fact., 9, 22 (2010).
39. Malki, A., Le, H. T., Milles, S., Kern, R., Caldas, T., Abdallah, J., and
Richarme, G.: Solubilization of protein aggregates by the acid stress chaper-
ones HdeA and HdeB, J. Biol. Chem., 283, 13679e13687 (2008).
40. Seo, J. H., Kang, D. G., and Cha, H. J.: Comparison of cellular stress levels and
green-fluorescent-protein expression in several Escherichia coli strains, Bio-
technol. Appl. Biochem., 37, 103e107 (2003).
41. Higgins, C. F.: ABC transporters: from microorganisms to man, Annu. Rev. Cell
Biol., 8, 67e113 (1992).
42. Li, Z., Leung, W., Yon, A., Nguyen, J., Perez, V. C., Vu, J., Giang, W., Luong, L. T.,
Phan, T., Salazar, K. A., and other 7 authors: Secretion and proteolysis of
heterologous proteins fused to the Escherichia coli maltose binding protein in
Pichia pastoris, Protein Expr. Purif., 72, 113e124 (2010).
43. Sun, P., Tropea, J. E., and Waugh, D. S.: Enhancing the solubility of recombi-
nant proteins in Escherichia coli by using hexahistidine-tagged maltose-binding
protein as a fusion partner, Methods Mol. Biol., 705, 259e274 (2011).
44. Richarme, G. and Caldas, T. D.: Chaperone properties of the bacterial peri-
plasmic substrate-binding proteins, J. Biol. Chem., 272, 15607e15612 (1997).
45. Ferenci, T.: Adaptation to life at micromolar nutrient levels: the regulation of
Escherichia coli glucose transport by endoinduction and cAMP, FEMS Microbiol.
Rev., 18, 301e317 (1996).
Please cite this article in press as: Han, M.-J., Exploring the proteomic characteristics of the Escherichia coli B and K-12 strains in different cellular
compartments, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.12.005
J. BIOSCI. BIOENG.,
46. Gilson, E., Rousset, J. P., Charbit, A., Perrin, D., and Hofnung, M.: malM, a new
gene of the maltose regulon in Escherichia coli K12. I. malM is the last gene of
the malK-lamB operon and encodes a periplasmic protein, J. Mol. Biol., 191,
303e311 (1986).
47. Stenberg, F., Chovanec, P., Maslen, S. L., Robinson, C. V., Ilag, L. L., von
Heijne, G., and Daley, D. O.: Protein complexes of the Escherichia coli cell
envelope, J. Biol. Chem., 280, 34409e34419 (2005).
48. Nikaido, H.: Molecular basis of bacterial outer membrane permeability revis-
ited, Microbiol. Mol. Biol. Rev., 67, 593e656 (2003).
49. Huang, C. Z., Lin, X. M., Wu, L. N., Zhang, D. F., Liu, D., Wang, S. Y., and
Peng, X. X.: Systematic identification of the subproteome of Escherichia coli cell
envelope reveals the interaction network of membrane proteins and
membrane-associated peripheral proteins, J. Proteome Res., 5, 3268e3276
(2006).
50. Maddalo, G., Stenberg-Bruzell, F., Götzke, H., Toddo, S., Björkholm, P.,
Eriksson, H., Chovanec, P., Genevaux, P., Lehtiö, J., Ilag, L. L., and Daley, D. O.:
Systematic analysis of native membrane protein complexes in Escherichia coli,
J. Proteome Res., 10, 1848e1859 (2011).
51. Terashima, H., Kojima, S., and Homma, M.: Flagellar motility in bacteria
structure and function of flagellar motor, Int. Rev. Cell Mol. Biol., 270, 39e85
(2008).
52. Archer, C. T., Kim, J. F., Jeong, H., Park, J. H., Vickers, C. E., Lee, S. Y., and
Nielsen, L. K.: The genome sequence of E. coli W (ATCC 9637): comparative
genome analysis and an improved genome-scale reconstruction of E. coli, BMC
Genomics, 12, 9 (2011).
53. Maurer, L. M., Yohannes, E., Bondurant, S. S., Radmacher, M., and
Slonczewski, J. L.: pH regulates genes for flagellar motility, catabolism, and
oxidative stress in Escherichia coli K-12, J. Bacteriol., 187, 304e319 (2005).
54. Minamino, T., Imae, Y., Oosawa, F., Kobayashi, Y., and Oosawa, K.: Effect of
intracellular pH on rotational speed of bacterial flagellar motors, J. Bacteriol.,
185, 1190e1194 (2003).
55. MacNab, R. M.: Flagella and motility, pp. 123e145, in: Neidhardt, F. C.,
Curtiss, I. R., II, Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B.,
Reznikoff, W. S., Riley, M., Shaechter, M., and Umbarger, H. E. (Eds.), Escherichia
coli and Salmonella typhimurium: cellular and molecular biology, 2nd ed. ASM
Press, Washington, DC (1996).
56. Sharma, S. S., Blattner, F. R., and Harcum, S. W.: Recombinant protein pro-
duction in an Escherichia coli reduced genome strain, Metab. Eng., 9, 133e141
(2007).
57. Herrera, G., Martinez, A., Blanco, M., and O’Connor, J. E.: Assessment of
Escherichia coli B with enhanced permeability to fluorochromes for flow
cytometric assays of bacterial cell function, Cytometry, 49, 62e69 (2002).
58. Schneider, D., Duperchy, E., Depeyrot, J., Coursange, E., Lenski, R., and
Blot, M.: Genomic comparisons among Escherichia coli strains B, K-12, and
O157:H7 using IS elements as molecular markers, BMC Microbiol., 2, 18
(2002).
59. Nandi, B., Nandy, R. K., Sarkar, A., and Ghose, A. C.: Structural features,
properties and regulation of the outer-membrane protein W (OmpW) of Vibrio
cholerae, Microbiology, 151, 2975e2986 (2005).
60. Francetic, O., Belin, D., Badaut, C., and Pugsley, A. P.: Expression of the
endogenous type II secretion pathway in Escherichia coli leads to chitinase
secretion, EMBO J., 19, 6697e6703 (2000).
61. Gentschev, I., Dietrich, G., and Goebel, W.: The E. coli alpha-hemolysin
secretion system and its use in vaccine development, Trends Microbiol., 10,
39e45 (2002).
62. Nandakumar, M. P., Cheung, A., and Marten, M. R.: Proteomic analysis of
extracellular proteins from Escherichia coli W3110, J. Proteome Res., 5,
1155e1161 (2006).
63. Jeong, K. J. and Lee, S. Y.: Excretion of human beta-endorphin into culture
medium by using outer membrane protein F as a fusion partner in recombinant
Escherichia coli, Appl. Environ. Microbiol., 68, 4979e4985 (2002).
64. Selkrig, J., Mosbahi, K., Webb, C. T., Belousoff, M. J., Perry, A. J., Wells, T. J.,
Morris, F., Leyton, D. L., Totsika, M., Phan, M. D., and other 12 authors:
Discovery of an archetypal protein transport system in bacterial outer mem-
branes, Nat. Struct. Mol. Biol., 19, 506e510 (2012).
65. McBroom, A. J. and Kuehn, M. J.: Release of outer membrane vesicles by gram-
negative bacteria is a novel envelope stress response, Mol. Microbiol., 63,
545e558 (2007).
66. Choi, D. S., Kim, D. K., Kim, Y. K., and Gho, Y. S.: Proteomics of extracellular
vesicles: exosomes and ectosomes, Mass Spectrom. Rev., 34, 474e490 (2015).
67. Zhang, G., Brokx, S., and Weiner, J. H.: Extracellular accumulation of recom-
binant proteins fused to the carrier protein YebF in Escherichia coli, Nat. Bio-
technol., 24, 100e104 (2006).
68. Majander, K., Anton, L., Antikainen, J., Lång, H., Brummer, M.,
Korhonen, T. K., and Westerlund-Wikström, B.: Extracellular secretion of
polypeptides using a modified Escherichia coli flagellar secretion apparatus,
Nat. Biotechnol., 23, 475e481 (2005).
69. Qian, Z. G., Xia, X. X., Choi, J. H., and Lee, S. Y.: Proteome-based identification
of fusion partner for high-level extracellular production of recombinant pro-
teins in Escherichia coli, Biotechnol. Bioeng., 101, 587e601 (2008).
VOL. xx, 2015
70. Daegelen, P., Studier, F. W., Lenski, R. E., Cure, S., and Kim, J. F.: Tracing
ancestors and relatives of Escherichia coli B, and the derivation of B strains
REL606 and BL21(DE3), J. Mol. Biol., 394, 634e643 (2009).
71. Xia, X. X., Qian, Z. G., Ki, C. S., Park, Y. H., Kaplan, D. L., and Lee, S. Y.: Native-
sized recombinant spider silk protein produced in metabolically engineered
Escherichia coli results in a strong fiber, Proc. Natl. Acad. Sci. USA, 107,
14059e14063 (2010).
72. Lee, P. S. and Lee, K. H.: Engineering HlyA hypersecretion in Escherichia coli
based on proteomic and microarray analyses, Biotechnol. Bioeng., 89, 195e205
(2005).
73. Han, M. J., Park, S. J., Park, T. J., and Lee, S. Y.: Roles and applications of small
heat shock proteins in the production of recombinant proteins in Escherichia
coli, Biotechnol. Bioeng., 88, 426e436 (2004).
74. Aldor, I. S., Krawitz, D. C., Forrest, W., Chen, C., Nishihara, J. C., Joly, J. C., and
Champion, K. M.: Proteomic profiling of recombinant Escherichia coli in high-
cell-density fermentations for improved production of an antibody fragment
biopharmaceutical, Appl. Environ. Microbiol., 71, 1717e1728 (2005).
75. Lee, S. H., Kang, K. H., Kim, E. Y., Chae, T. U., Oh, Y. H., Hong, S. H., Song, B. K.,
Jegals, J., Park, S. J., and Lee, S. Y.: Metabolic engineering of Escherichia coli for
enhanced biosynthesis of poly(3-hydroxybutyrate) based on proteome anal-
ysis, Biotechnol. Lett., 35, 1631e1637 (2013).
76. Song, J. A., Lee, D. S., Park, J. S., Han, K. Y., and Lee, J.: A novel Escherichia coli
solubility enhancer protein for fusion expression of aggregation-prone heter-
ologous proteins, Enzyme Microb. Technol., 49, 124e130 (2011).
77. Han, K. Y., Seo, H. S., Song, J. A., Ahn, K. Y., Park, J. S., and Lee, J.: Transport
proteins PotD and Crr of Escherichia coli, novel fusion partners for heterologous
protein expression, Biochim. Biophys. Acta, 1774, 1536e1543 (2007).
Please cite this article in press as: Han, M.-J., Exploring the proteomic characteristics of the Escherichia coli B and K-12 strains in different cellular
compartments, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.12.005
PROTEOMIC COMPARISON OF E. COLI B AND K-12 9
78. Kang, Y. S., Song, J. A., Han, K. Y., and Lee, J.: Escherichia coli EDA is a novel
fusion expression partner to improve solubility of aggregation-prone heterol-
ogous proteins, J. Biotechnol., 194, 39e47 (2015).
79. Park, J. S., Han, K. Y., Song, J. A., Ahn, K. Y., Seo, H. S., and Lee, J.: Escherichia
coli malate dehydrogenase, a novel solubility enhancer for heterologous pro-
teins synthesized in Escherichia coli, Biotechnol. Lett., 29, 1513e1518 (2007).
80. Park, J. S., Han, K. Y., Lee, J. H., Song, J. A., Ahn, K. Y., Seo, H. S., Sim, S. J.,
Kim, S. W., and Lee, J.: Solubility enhancement of aggregation-prone heter-
ologous proteins by fusion expression using stress-responsive Escherichia coli
protein, RpoS, BMC Biotechnol., 8, 15 (2008).
81. Han, K. Y., Song, J. A., Ahn, K. Y., Park, J. S., Seo, H. S., and Lee, J.: Solubilization
of aggregation-prone heterologous proteins by covalent fusion of stress-
responsive Escherichia coli protein, SlyD, Protein Eng. Des. Sel., 20, 543e549
(2007).
82. Han, K. Y., Song, J. A., Ahn, K. Y., Park, J. S., Seo, H. S., and Lee, J.: Enhanced
solubility of heterologous proteins by fusion expression using stress-induced
Escherichia coli protein, Tsf, FEMS Microbiol. Lett., 274, 132e138 (2007).
83. Han, M. J., Lee, J. W., Lee, S. Y., and Yoo, J. S.: Proteome-level responses of
Escherichia coli to long-chain fatty acids and use of fatty acid inducible pro-
moter in protein production, J. Biomed. Biotechnol., 2008, 735101 (2008).
84. Xia, X. X., Qian, Z. G., and Lee, S. Y.: Comparative proteomic and genetic an-
alyses reveal unidentified mutations in Escherichia coli XL1-Blue and DH5a,
FEMS Microbiol. Lett., 314, 119e124 (2011).
85. Yim, S. S., An, S. J., Han, M. J., Choi, J. W., and Jeong, K. J.: Isolation of a po-
tential anchoring motif based on proteome analysis of Escherichia coli and its
use for cell surface display, Appl. Biochem. Biotechnol., 170, 787e804 (2013).