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Journal of Bacteriology, January 2001, p. 545-556, Vol. 183, No. 2
Central Research and Development, DuPont
Company, Wilmington, Delaware 19880-01731;
Agricultural Products, DuPont Company, Newark, Delaware
19714-61042; and Department of
Genetics, University of Wisconsin, Madison, Wisconsin 537063
Received 5 September 2000/Accepted 25 October 2000
A nearly complete collection of 4,290 Escherichia coli
open reading frames was amplified and arrayed in high density on glass slides. To exploit this reagent, conditions for RNA isolation from
E. coli cells, cDNA production with attendant fluorescent dye incorporation, DNA-DNA hybridization, and hybrid quantitation have
been established. A brief
isopropyl- Escherichia coli K-12 has
been exhaustively studied for over 50 years. Early experiments measured
the molecular fluxes from small compounds into macromolecular
constituents (33). These studies were followed by others
in which small molecule pools of central metabolic building blocks
(21), nucleotides (3), and amino acids were
enumerated. The levels of several macromolecular components, including
individual species of proteins (26), have been measured.
Such measurements of the steady state provide a census of the cellular
content, while changes upon imposition of a stress catalogue the
cell's fight for survival. This response to an insulting or adverse
condition can take many forms, from relieving end product inhibition to
derepressing transcription (20).
In E. coli, experiments to define stress-related, global
regulatory responses have often relied upon either the isolation of
operon fusions induced by a particular stress (16)
or proteomic measures in which the protein fractions from stressed and
unstressed cultures are separated by a two-dimensional method prior
to comparison (37). Each method has an inherent
technological hurdle; the map location of responsive gene fusions must
be ascertained precisely, while induced or repressed proteins excised
from the two-dimensional gels must be correctly identified.
Alternatively, mRNA measurements utilizing techniques such as
hybridization to DNA and primer extension have allowed the monitoring of individual gene's expression profiles. Recently, expression profiling of most yeast genes has been reported (8, 40); such measurements were facilitated by high-density arrays of individual genes and specific labeling of cDNA copies of eukaryotic mRNA by using
poly(A) tail-specific primers. Thus, the lack of a poly(A) tail and the
extremely short bacterial mRNA half-life represent hurdles for the
application of DNA microarray technology to prokaryotic research.
Nonetheless, early attempts at comprehensive expression profiling using
large DNA fragments from an ordered Here we present a means to successfully perform microarray-based
comprehensive gene expression profile analyses with E. coli. We show that such experimentation can be informative by examination of
(i) differences in gene expression profiles caused by growth of
E. coli in either minimal or rich medium, (ii) changes in
gene expression associated with the transition from exponential-phase to stationary-phase growth in minimal medium, and (iii) the specificity of induction mediated by
isopropyl- Microbiological methods.
E. coliMG1655
(1) was cultured with aeration in either the minimal
medium, M9 (23), supplemented with 0.4% glucose or in the
rich medium, Luria-Bertani (LB) (23), at 37°C. The
overnight culture was diluted 250-fold into fresh medium and aerated at 37°C. Samples of the minimal medium culture were harvested at A600s of 0.40 (exponential phase; just under
five generations) and 1.6 (transition to stationary phase, just less
than seven generations) prior to RNA isolation. An IPTG induction
(23) was performed to examine the specificity with which
it affects gene expression. The LB medium-grown culture was split when
it achieved an appropriate density (A600 of
0.40). To one portion was added IPTG to a final concentration of 1 mM;
the untreated sample served as a control. Incubation of both samples
was continued with aeration at 37°C for another 15 min
(A600 of 0.45 for both cultures) before RNA
isolation was initiated.
RNA isolation.
Shaved ice was added to 50-ml samples which
were pelleted immediately in a refrigerated centrifuge by spinning at
10,410 × g for 2 min. Each resultant pellet was
resuspended in a mixture containing 100 µl of Tris-HCl (10 mM, pH
8.0) and 350 µl of Synthesis of fluorescent cDNA from total RNA.
To a volume
brought to 22 µl with double-distilled water (ddH2O) were
added 6 µg of total RNA template and 12 µg of random hexamer
primers (Operon Technologies, Inc., Alameda, Calif.). Annealing was
accomplished by incubation for 10 min at 70°C followed by 10 min at
room temperature. cDNA probes were synthesized with SuperScript II
reverse transcriptase (10 U/µl; Life Technologies, Inc.,
Gaithersburg, Md.) in the presence of deoxynucleoside triphosphates (dNTPs) (dATP, dGTP, and dTTP, each at 0.1 mM; dCTP at 50 µM) and
Cy3- or Cy5-dCTP at 25 µM. In order were added 8 µl of 5× SuperScript II reaction buffer (Life Technologies, Inc.), 4 µl of 0.1 M dithiothreitol, 2 µl of the dNTP mix (2 mM dATP, 2 mM dGTP, 2 mM
TTP, 1 mM dCTP), 2 µl of 0.5 mM Cy3- or Cy5-labeled dCTP (Amersham
Pharmacia Biotech, Arlington Heights, Ill.), and 2 µl of SuperScript
II reverse transcriptase. cDNA synthesis proceeded at 42°C for
2.5 h before the reaction was terminated by heating at
94°C for 5 min. The RNA templates were hydrolyzed with 0.25 M NaOH.
The reaction was then neutralized by adding HCl and Tris-HCl (pH 6.8).
The labeled cDNA was purified with a PCR purification kit (Qiagen),
dried, and stored at Fluorescent copying of genomic DNA.
Genomic DNA was isolated
from strain MG1655 by a standard procedure (38). Genomic
DNA, sheared with a nebulizer to approximately 2-kbp fragments, was
used to prepare labeled DNA. Three micrograms of this DNA was mixed
with 6 µg of random hexamer primers (Operon Technologies, Inc.) in 33 µl of ddH2O. DNA was denatured by heating at 94°C prior
to annealing on ice for 10 min. Fluorescent copying of the genomic DNA
was accomplished with the Klenow fragment of DNA polymerase I (5 U/µl; Promega, Madison, Wis.). To the DNA mixture was added 6 µl of
10× Klenow buffer (supplied with the enzyme), 3 µl of the dNTP mix
described above, 12 µl of ddH2O, 3 µl of 0.5 mM
Cy3-dCTP (Amersham Pharmacia Biotech), and 3 µl of the Klenow
fragment of DNA polymerase I. After a static, 2.5-h incubation at room
temperature, the labeled DNA probe was purified with a PCR purification
kit (Qiagen) before being dried in a speed vacuum.
Amplification of 4,290 E. coli genes.
Our
amplification method was based on a previously described protocol
(31). Specific primer pairs (Sigma Genosys, The Woodlands, Tex.) for each protein-specifying gene of E. coli were used
in two consecutive PCR amplifications. Two amplifications were
performed to prevent contaminating genomic DNA within the initial PCR
product from being spotted to the microarray. Any such carried-over
material was eliminated by the "dilution" associated with the
second amplification reaction. Genomic DNA (30 ng) was used as the
template in the first round of PCR amplification, and 500-fold-diluted
PCR products served as templates for PCR reamplification. Duplicate
50-µl scale reactions were performed in the reamplification. The PCRs
were catalyzed with ExTaq polymerase (Panvera, Madison, Wis.) with the
four dNTPs (Amersham Pharmacia Biotech) present at 0.2 mM and the
primers at 0.5 µM. Twenty-five cycles of denaturation at 95°C for
15 s, annealing at 64°C for 15 s, and polymerization at 72°C
for 1 min were conducted. A 2-µl aliquot of each PCR product was
sized by electrophoresis through agarose gels. More than 95% of these
resultant second PCR products displayed visible bands of the correct
size. Second-round PCR mixtures, devoid of templates and primers, were
saved to be spotted onto slides to serve as negative controls for
hybridization experiments. Each second-round PCR mixture was purified
with 96-well PCR purification kits (Qiagen). The eluant was dried with
a vacuum centrifuge.
Arraying amplified genes.
Twenty microliters of 6 M
Na2SCN or 50% dimethyl sulfoxide was added to each dried
DNA sample ( Hybridization and washing.
Arrayed slides were placed in
isopropanol for 10 min, boiled in ddH2O for 5 min, and
dried by passage of ultraclean N2 gas prior to
prehybridization. The prehybridization solution (PHS) was 3.5× SSC
(1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) (BRL, Life
Technologies Inc., Gaithersberg, Md.), 0.2% sodium dodecyl sulfate
(SDS; BRL, Life Technologies, Inc.), 1% bovine serum albumin (fraction
V; Sigma, St. Louis, Mo.). The hybridization solution (HS) contained 4 µl of ddH2O, 7.5 µl of 20× SSC, 2.5 µl of 1% SDS
(BRL, Life Technologies Inc.), 1 µl of 10 mg of salmon sperm DNA per
ml (Sigma), and 15 µl of formamide (Sigma). The slides were incubated
at 60°C for 20 min in PHS to block nonspecific binding of probe. The
slides were next rinsed five times in ddH2O at room
temperature and twice in isopropanol before being dried by the passage
of nitrogen. The dried probe was resuspended in the HS and denatured by
heating at 94°C for 5 min. Thirty microliters of the probe containing
HS was applied to a dried, prehybridized slide, covered with a
coverslip (Corning, Corning, N.Y.), and put into a sealed hybridization
chamber containing a small reservoir of water to maintain moisture.
Hybridization occurred for approximately 14 h at 35°C.
Coverslips were removed in washing buffer I (2× SSC-0.1% SDS) warmed
to 35°C prior to incubation for 5 min. Next, the slides were washed
sequentially for 5 min in 1× SSC-0.1% SDS and 0.1× SSC-0.1% SDS.
Slides were then passed through three baths, each passage lasting 2 min, of 0.1× SSC. The slides were dried with a nitrogen gas flow.
Data collection and analysis.
Hybridization to each slide
was quantified with a confocal laser microscope (Molecular Dynamics,
Sunnyvale, Calif.) the photomultiplier tube of which was set to 700 and
800 V for the Cy-3 and Cy-5 signals, respectively. The images obtained
were analyzed with ArrayVision 4.0 software (Imaging Research, Inc.,
Ontario, Canada). The fluorescent intensity (Ii)
associated with each spotted gene (i) was reduced by
subtracting the fluorescence (Ni) of an
adjoining, nonspotted region of the slide. These readings
(Ri = Ii
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.545-556.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
High-Density Microarray-Mediated Gene
Expression Profiling of Escherichia coli
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Appendix
References
-D-thiogalactopyranoside (IPTG) treatment
elevated lacZ, lacY, and lacA
transcript content about 30-fold; in contrast, most other transcript
titers remained unchanged. Distinct RNA expression patterns between
E. coli cultures in the exponential and transitional phases
of growth were catalogued, as were differences associated with
culturing in minimal and rich media. The relative abundance of each
transcript was estimated by using hybridization of a genomic
DNA-derived, fluorescently labeled probe as a correction factor. This
inventory provided a quantitative view of the steady-state level of
each mRNA species. Genes the expression of which was detected by this
method were enumerated, and results were compared with the current
understanding of E. coli physiology.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Appendix
References
library of E. coli
genomic fragments as a capture reagent and radiolabeled cDNA as a probe
suggested that these problems were not insurmountable (6).
-D-thiogalactopyranoside (IPTG), the classic
lac operon inducer. Moreover, a method for
determining the relative abundance of each transcript was developed and
used to provide a census of the mRNA composition of E. coli
under each of the growth conditions mentioned above.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Appendix
References
-mercaptoethanol-supplemented RLT buffer
(Qiagen RNeasy Mini kit; Valencia, Calif.) that was kept on ice. The
cell suspension was added to a chilled 2-ml microcentrifuge tube
containing 100 µl of 0.1-mm-diameter zirconia-silica beads (Blospec
Products Inc., Bartlesville, Okla.). The cells were broken by agitation
at room temperature for 25 s with a Mini-Beadbeater (Biospec
Products, Inc.). Debris was pelleted by centrifugation for 3 min at
16,000 × g and 4°C; the resultant supernatant was mixed with 250 µl of ethanol. This mixture was loaded onto Qiagen RNeasy columns from the Qiagen RNeasy Mini kit. RNA isolation was
completed by using the protocol supplied with this kit. Incubation for
1 h at 37°C in 40 mM Tris (pH 8.0), 10 mM NaCl, 6 mM
MgCl2 with RNase-free RQ1 DNase (1 U/µl; Promega,
Madison, Wis.) digested any genomic DNA contaminating the RNA
preparation. The digestion products were purified by a second passage
through an RNeasy column (Qiagen). The product was eluted from the
column in 50 µl of RNAse-free water prior to determining sample
concentration by an A260 reading. RNA
preparations were stored frozen at 
20°C until use.
20°C. Labeling efficiency was calculated by
using the A260 and either
A550 for Cy3 incorporation or A650
for Cy5 labeling measurements.
0.1-ng/nl final concentration). A generation II DNA
spotter (Molecular Dynamics, Sunnyvale, Calif.) was used to array the
samples onto coated glass slides (Amersham Pharmacia Biotech). Two
aliquots of approximately 1 nl from 1,536 resuspended PCR products were
arrayed in duplicate on each slide; three slides were used to order all
amplified E. coli genes. To serve as controls, 76 specific
E. coli PCR products, 8 amplified genes of Klebsiella
pnuemoniae, and 12 plant cDNA clones were also spotted onto each
slide. Arrayed glass slides, after baking at 80°C for 2 h, were
stored in a desiccator at room temperature under vacuum.
Ni) were exported to a spreadsheet for further
data manipulation. The four "no-DNA" spots derived from PCR
mixtures devoid of template were controls used to determine the noise
(background signal) level.
in ER = ERi); normalization by multiplication with a
correction factor (CF) of the summed values and the underlying
equivalent readings was performed to equalize the summed readings of
the control and treated samples
(in ERcontrol = CF × in
ERtreated). This allowed calculation of fold induction of
each gene's expression by comparison of each gene's normalized
equivalent reading, norm ER, from a pair of conditions. The fold
induction of any gene's transcript by a chemical treatment is norm
ERtreated/ norm ERcontrol, where norm
ERcontrol = ERcontrol and norm
ERtreated = CF × ERtreated.
RNA abundance. To convert normalized equivalent readings into measures of transcript abundance (AB), a further correction was needed. That correction required the hybridization signal arising from an equimolar concentration of all transcripts. The surrogate for this correction factor was the fluorescent intensities arising from hybridization with the fluorescent copy of genomic DNA. Thus, the fluorescent intensities from hybridization with RNA-derived probes were corrected by using fluorescent intensities arising from genomic DNA-derived probes. The abundance of each gene's transcription product(s) was determined by dividing the normalized equivalent reading of a genomic DNA-derived sample into the normalized equivalent reading from the RNA-derived sample (AB = norm ERtranscripts/ norm ERgenome). The convention of Riley and Labedan (32) was followed in grouping genes into functional sets.
-Galactosidase content.
-Galactosidase content
was measured by the method of Miller using the combined action of
chloroform and SDS to disrupt the cell envelope, thus allowing entry of
the substrate (23). Two independent cultures of MG1655
were grown in LB medium at 37°C to densities
(A600s) of 0.43 and 0.47, split, treated with 1 mM IPTG for 15 min, and assayed. The data from these two independent experiments performed on different days were averaged.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Appendix
References
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Array quality. Preliminary experiments (data not shown) indicated that the fluorescent signals obtained with labeled cDNA derived from random priming of E. coli genomic DNA (3 µg) were not saturating and were well within the linear range of the instrumentation. The noise level was determined by averaging the readings of the four control spots derived from PCR mixtures lacking a DNA template after subtracting the background signal derived from an adjacent area that had not been spotted.
Using a probe derived from genomic DNA, hybridization signals exceeding noise by a factor of 2 were observed for 4,228 (99.5%) of the 4,290 arrayed genes. Sixty two genes (see Appendix) either failed in PCR amplifications or did not bind sufficient signal to be detected when present at a presumably equimolar ratio with the other genes in the probe sample. Interestingly, 4 (ilvL, leuL, rhoL, and tnaL) of the 27 known genes of this class encode short (<300 bp), attenuation leader polypeptides (19). This suggests that short open reading frames ORFs may not be as readily detected with this method as longer genes.IPTG induction.
The effect of 1 mM IPTG upon expression of the
arrayed genes was investigated. Duplicate RNA preparations of the
control and induced cells were each labeled with Cy-3 and Cy-5 by
cDNA synthesis. Averaging of measurements was essential for optimal
signal detection (Fig. 1).
lacZYA induction above the background was detected when the
results of a single hybridization experiment in which Cy3-labeled cDNAs derived from treated and control cells were separately
hybridized to individual slide sets as viewed in a log-log plot (Fig.
1A). Variation in the measurement of other transcripts was also
significant, as indicated by the width of the spread in the data points
falling along the diagonal of this scatter plot. Thus, dual-labeling
experiments were performed. Improvements were observed by labeling the
control sample with Cy-5 and the induced sample with Cy-3 before
hybridizing to a single set of three slides (Fig. 1B). However, there
was a skewing of the data away from the abscissa (x axis)
and towards the ordinate (y axis).
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-galactosidase
content of MG1655 cultures treated with 1 mM IPTG for 15 min resulted
in 540 ± 60 Miller units (n = 2), while untreated
cultures (n = 2) yielded 17 ± 9 Miller units. Thus, lac transcription and translation increased in
parallel.
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-galactosidase, while uxaA
specifies an enzyme of hexuronate catabolism (2). Interestingly, potential melA induction by IPTG was
suggested by hybridization of cDNA to an ordered set of clones
carrying inserts of the E. coli chromosome (6)
and in another microarray experiment (31).
The function of the other induced genes is even more speculative;
upstream of b1783 is a 
54 binding site, while
peptidase function is hypothesized for b2324. Induction of
these latter genes was not observed by Richmond et al.
(31).
Estimate of steady-state transcript levels. The percentage of RNA that programs protein synthesis has been determined under a wide variety of growth regimens (4). Here we estimated the fraction of those protein-specifying transcripts devoted to each arrayed gene. Hybridization signals arising from annealing of RNA-derived Cy-3 labeled cDNA populations were normalized by dividing by the signal generated with Cy-3 fluorescent cDNA arising from copying of sheared E. coli genomic DNA as a probe. Each spot's corrected signal from RNA-derived cDNA hybridization reflected the amount of RNA in the sample. Three RNA samples were thus measured; they were isolated from cells growing exponentially in rich medium, growing exponentially in minimal medium, and cells in minimal medium making a transition from the exponential phase to the stationary phase (for culture conditions, see Materials and Methods). RNAs from certain central metabolic (gapA and ptsH), defense (ahpC and cspC), DNA metabolic (hns), surface structure (acpP, ompACFT, and lpp), translation (rplBCKLMPWX, rpmBCl, rpsACDHJNS, trmD, fusA, infC, and tufAB), transcription (rpoAB), and unassigned (b4243) genes (32) were abundant (>0.1%, among the top 100 transcripts) in all three samples.
Transcripts of exponential-phase cells cultured in rich medium. High-density microarrays were used to measure the transcriptional content of cells growing in rich medium. A total of 1,776 genes were apparently not expressed; their hybridization signals did not exceed the noise by a factor of 2. Of these nonexpressed genes, function has been ascribed to 465. They are listed in the Appendix. Each gene having an assigned function was expressed in at least one of the two tested stages of cultivation in minimal medium.
The other genes, each representing between 0.0007 and 1% of the summed hybridizing signal, were expressed in LB broth-grown cells. The distribution of genes as a function of expression level is plotted in Fig. 2, while Fig. 3 depicts fractional expression as a function of summed genes. The percentage of transcripts was plotted as a function of genes summed in Fig. 3. The order in which genes were summed was based upon expression level, with the most highly expressed gene in each condition summed first.
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), S15, L35, S21 (), and L13
operons. The bases of such gradients were not investigated.
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Transcripts of exponential-phase cells cultured in defined minimal medium. The gene expression pattern of cells growing exponentially in minimal medium was also examined. At this cell density, the pH remained at 7.0. Expression levels varied from 0.001 to 0.7%. Apparently, as illustrated in Fig. 2, biosynthetic requirements mandated that a significantly greater fraction of the genome was expressed in minimal medium. Only 776 genes were expressed negligibly (signal/noise ratio of <2); 149 of these genes have presumed or demonstrated function. They are listed in the Appendix.
The 50 genes most highly expressed in logarithmically growing cells cultured in minimal medium with glucose as a carbon or energy source are enumerated in middle columns of Table 2. The distribution of genes as a function of expression level (Fig. 2) and the fractional expression as a function of summed genes with genes ranked by expression level (Fig. 3) were also plotted. Such broad-distribution analyses readily revealed the significant differences observed in expression of E. coli when grown in defined and rich media. In minimal media, many more genes were transcribed over a somewhat broader range. Eight biosynthetic genes became highly expressed (Table 2). Notable among them were metE, encoding the aerobic methionine synthase, and ilvC, an isoleucine-valine biosynthetic gene subject to feed-forward transcriptional activation (35) by its substrates. Both the ilvC (27, 39)- and metE (10)-encoded enzymes are sluggish catalysts. The metE product accounts for about 5% of E. coli protein when cells are cultured in minimal medium with glucose as a carbon or energy source (36). Other highly expressed biosynthetic genes included folE and cysK;the folE product, GTP cyclohydrolase I, catalyzes both cleavage of the 5-membered ring of guanine and the rearrangement of the ribose moiety of the substrate, GTP (9). cysK, encoding o-acetylserine (thiol)-lyase isozyme A, is responsible for more than 90% of sulfur fixation under aerobic conditions (17). Transcripts of the pyrBI operon encoding aspartate transcarbamylase also were highly expressed during exponential growth in minimal medium relative to an LB broth-grown culture. This expression level is a characteristic signature of strain MG 1655, whose aspartate transcarbamylase content is elevated more than 100-fold when grown in the absence of uracil due to an rph mutation that is polar on pyrE (13). The other highly expressed transcripts, thrL and aroF, encoded, respectively, the threonine leader polypeptide (19) and the phenylalanine-inhibited first enzyme of the common aromatic pathway. The aroF product, one of three isozymes, is estimated to account for more than 80% of the activity catalyzing the first common step of aromatic amino acid synthesis (28). Expression of several genes catalyzing fueling reactions was also elevated. As expected, ptsHI transcripts encoding phosphotransferase sugar transport common components (29) accumulated to a very high titer in glucose-minimal medium. Surprisingly, aceAB, encoding the glyoxylate shunt enzymes malate synthase and isocitrate lyase (7), was highly expressed. Perhaps the tricarboxylic acid (TCA) cycle functions in its branched state during this phase of growth requiring the glyoxylate shunt for anapleurotic replenishment (24). Alternatively, at this culture density, the cells have started to use the accumulated acetate. These data were further analyzed by examining individual transcript levels within the context of operon structure (Table 4). Results with selected amino acid biosynthetic operons are presented in conjunction with the operon responsible for phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS)-mediated sugar uptake. These biosynthetic operons are predominantly controlled by attenuation. Detection of attenuator transcripts was difficult (see above and the Appendix) this difficulty was compounded by the multiple PCR products obtained after amplification of hisL (Table 4). The thr leader was found to be more highly expressed than the cognate structural genes; this was not observed when the trp or his operons were analyzed. The most poorly expressed of the listed genes was ilvY, which encodes a specific DNA binding protein that activates ilvC transcription while repressing its own synthesis (35). Conversely, ilvC was the most highly expressed of this group, as was indicated in Table 2. Measurement of pts operon expression ranged from 1,200 to 3,100 ppm; a gradient of transcript levels was observed for pts. Multiple promoters and termination sites, a hallmark of this operon (29), might provide a basis for this observation.
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Transcripts of cells making a transition from the exponential phase to the stationary phase in defined minimal medium. During this transition, at a point where the pH had dropped slightly to 6.7, significant changes in gene expression were expected and observed. Expressed gene levels were from 0.0023 to 1.6%. A total of 1,030 genes, of which 110 have a defined role (Appendix) did not appear to be expressed at this transitional phase of growth.
The 50 genes most highly expressed genes during this transition are listed in the rightmost columns of Table 2. Significantly, several rpoS-regulated genes, including hdeA (10-fold transcript elevation in comparison to the exponential-phase content) (12), hdeB (9-fold) (12), dps (4-fold) (12), gadA (8-fold) (5), and gadB (10-fold) (5), as well as rpoS (3-fold) (12) itself, became highly expressed. Despite this remodeling of transcription, the overall patterns of gene number as a function of expression level (Fig. 2) and fractional expression as a function of ranked gene (Fig. 3) were not as distinct as one might imagine in comparison to the patterns from exponentially growing cells.Compilation.
The observed expression patterns are summarized
in Table 5, where gene
products were grouped by metabolic function according to an established
classification scheme (32). Exponential growth in
minimal medium elevated the amount of pyrimidine and amino acid
biosynthetic transcripts with respect to growth in the rich broth, LB.
In contrast, cofactor and purine transcripts did not appear to
accumulate relative to growth in LB broth. Expression of glyoxylate
shunt and other glucose metabolism-related transcripts was also
elevated in minimal medium; the seven-fold elevation of glyoxylate
shunt transcripts exceeded the average of that observed for amino
acid biosynthetic mRNAs. Expression of genes involved in
sulfur fixation was also elevated during growth in minimal medium.
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S-dependent fashion, serves
as an osmoprotectant (11). The cell also displayed an
increased titer of protein folding and global regulatory function
transcripts while making a transition between growth phases.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Appendix
References
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Comprehensive expression profiling has been performed previously with the yeast Saccharomyces cerevisiae (8). Here we report that such profiling can also be accomplished and refined by using a dye-swapping, high-density microarray technology when the prokaryote E. coli is used as an experimental system. Adaptation of RNA isolation and labeling protocols from eukaryotes to prokaryotes is not straightforward, because eukaryotic mRNA manipulations often exploit the specific 3'-polyadenylation of this molecular species. The short half-life of bacterial mRNA is another obstacle. We chose to isolate RNA by a standard procedure that included a centrifugation step and to reverse transcribe bulk prokaryotic RNA to prepare our hybridization probe. Thus, the reported measurements are subject to possible systematic errors, including differential mRNA stabilities (18). Despite the large amount of stable RNA in the sample, hybridization to protein-encoding genes was readily detected. Recently, independent studies of E. coli (31, 34) successfully applied nylon-based medium-density DNA array or glass-based high-density DNA array technologies to assess gene expression changes in response to growth medium and heat shock.
Nonetheless, errors could be introduced in the many steps from RNA purification to analyses of hybridization signals. As shown in Fig. 1, conditions have been optimized to yield highly reproducible data. The scatter plot of the optimized protocol (Fig. 1E) illustrated that measurements of gene expression were still subject to considerable variation when the signal was in the lowest part of the detectable range. It was found that expression of only eight genes was effected by IPTG treatment; all were induced. It was reassuring that the expected lacZYA induction was observed; the significance of the weaker inductions awaits confirmation by complementary techniques, perhaps based upon enzyme assay of gene fusions or analysis of an isogenic pair of strains differing in lacI. Such a correlation has been provided for IPTG induction of melA expression (31). Thus, such comprehensive gene expression profiling generates hypotheses requiring further study for verification.
Having developed confidence in the technology, it was applied to monitoring expression as a function of growth stage and medium. For these experiments, normalization of signal intensity was essential. Probe, derived from replication of genomic DNA and used as a replica of equimolar transcription of the entire genome, allowed calculation of mRNA inventories. Thus, we have provided measures of steady-state transcript levels under prescribed sets of conditions rather than the fold change in mRNA titers that represents the difference in expression between two conditions. These inventories were satisfying in several ways. First, the most highly transcribed genes in actively growing cells cultured in LB medium often encoded proteins involved in translation. In contrast, cultures at a similar growth stage in glucose minimal medium expressed to a very high level several small molecule biosynthetic genes and the means to utilize glucose.
Thus, agreement between this molecular analysis and the accumulated understanding of E. coli physiology (24, 25) was observed. This agreement was underscored in the analysis of cells making a transition from the exponential growth phase to the stationary phase; the elevated expression of several rpoS-controlled genes corresponded to expectations. Nonetheless, some caution is necessary; potential effects of differential mRNA stability (18) have yet to be considered.
It is most unlikely that the technology is limited to highly expressed genes. First, reproducible expression measurements were obtained over a wide dynamic range (Fig. 1E). Second, the data from Fig. 3 and Table 1 illustrate that lac operon expression, although low before IPTG induction, was detected, suggesting that most transcripts can be readily measured by the techniques described. The lower limits of expression that can be observed may be defined by the analyses of well-characterized "promoter-down" mutants (30) or "spiking" experiments. In the latter, the templates for cDNA synthesis would contain constant amounts of total RNA derived from a deletion mutant to which various quantities of a corresponding transcript synthesized in vitro has been added.
Compiling of data (Table 5) into functional groups (32) has become one method for the analysis of gene expression profiles. Work such as that presented here indicates that these categories do not respond as a bloc; rather subsets act differently, as has been observed for amino acid biosynthesis during a study of yeast (14). Nonetheless, such analyses allow global trends to be observed and the integration of gene expression patterns with the cell's overall physiological status. The histogram (Fig. 2) allows one to appreciate the quantity of transcripts that falls within each expression range. Figure 3 provides an indication of how transcriptional capacity is distributed under the three distinct conditions that were examined.
Moreover, unexpected results worthy of further study are found within the compilation presented in Table 5. Unlike transcripts dealing with energy transfer, ribosomal proteins, translation and aminoacyl-tRNA formation which were elevated in LB broth-grown cells, the sum of mRNAs specifying cell division proteins did not vary under the three conditions that were investigated. In a similar vein, hybridization to genes involved in the synthesis of the cell envelope was not increased when the probe was derived from cells cultured in LB broth.
In such global analyses, the reliability of the data obtained is an issue. The method described here makes four measurements of the transcript level present in each RNA sample. Moreover, the organization of genes into operons provides internal benchmarking of the measurements. Analyses of transcripts from ribosomal protein, biosynthetic, and PTS operons (Tables 3 and 4) suggests that the data are of high quality, since the range of the measurements for each operon is rather small. Such an analysis is consistent with the methodological improvements illustrated in Fig. 1.
Thus, an initial mRNA inventory was compiled. We believe that our analyses are subject to errors in measures of transcripts smaller than about 300 nucleotides and failures in PCR amplification. The compilation illustrated several physiological points. The protein biosynthetic demand during growth in rich medium was noted, accounting for about 15% of the polypeptide-specifying transcripts; what may be as significant is that more than one-quarter of all protein-specifying transcripts under any of the measured conditions lack a functional assignment. Consequently, gene expression profiling provides a further impetus to the continued study of E. coli.
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APPENDIX |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Appendix
References
|
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E. coli genes not giving a detectable signal when hybridized with genome-derived DNA were as follows: acrB, agaC, arcB, cydA, dacB, dnaT, entC, entF, exo, fruL, ilvL, leuL, lytB, pheL, phnA, potH, potl, putA, rbsD, rfaB, rhlB, rhoL, sdhC, selB, tdcA, tnaL, b0177, b0250, b0269, b0271, b0291, b0322, b0574, b1437, b1595, b1824, b1978, b2067, b2086, b2088, b2097, b2270, b2292, b2630, b2641, b2851, b2878, b3194, b3596, b3597, b3672, b3678, b3696, b3697, b3705, b4002, b4253, b4280, b4404, and b4405
Genes having a known function but not expressed when cells were cultured in rich medium were as follows: aas, acpDS, acrF, adiY, agaABDIRSVW, ais, alkB, alpA, apaGH, appY, aqpZ, araBEH, argACT, aroDE, arp, arsCR, artIMQ, asr, betIT, bglBG, bioCH, blc, bolA, cadAC, caiBF, cbl, cchB, ccmABCD, cdd, celAC, chaB, cheBRWYZ, chpAR, cmtAB, cof, cpsG, creB, criR, csgABDFG, cspBF, cvpA, cybC, cynST, cysHJUW, dam, dedA, deoR, dgkA, dicBC, dinIJ, dmsBC, dniR, dppBC, dsbE, dsdCX, dsrB, eaeH, ebgC, ecpD, emrY, endA, entDE, envRY, erfK, evgAS, exbB, farR, fdnI, fecIR, feoA, fepBDE, fes, fic, fimD_1, fimFGZ, fixX, flgACGHLMN, flhAD, fliACEFGJLMOPQRSTZ, folP, frdC, frvARX, frwD, ftsKL, fucAKORU, fumC, gabP, gadB, galKMPR, gapC, gatR, gcl, gcvA, gefL, gidB, gip, glcCDG, glgS, glnBK, glpEG, gltBDF, glvBCG, gntKUV, grxA, gusC, gutM, hdeAB, hdhA, hemH, hha, hhoB, hlpB, hlsMQ, hnr, hofBDFGH, holBE, hrpA, hslJS, htrCE, hyaBCDE, hybBDEFG, hycABDFGHI, hydN, hypACD, ibk, icc, ilvMNY, insA_2, insA_3, insA_4, katE, kch, kdgT, hdpE, kdtB, kduDI, kefC, lacA, lar, ldcC, leuDO, lhr, lit, livK, lrp, lysAR, marBR, may, mcrA, mdlB, mepA, metACR, mhpBE, moaD, moeB, molR, mreD, mscL, msyB, mukB, mutHY, nac, napBCFH, narIJZ, nel, nhaR, nikBDE, nirCD, nlp, nlpC, nrdEFG, nrfBFG, nupG, ogrK, osmBCE, pabA, panF, pfkB, pgpB, pheM, phnBCDFGHJKLOQ, phoBH, phpB, phrB, pinO, pitB, pnuC, potFG, ppdABCD, ppiC, prfH, priC, prkB, prmA, proBVW, prsA, pshM, psiF, pspBC, pssR, pstA, pth, ptrB, ptsO, purE, pyrIL, racC, rarD, rcsA, recDT, relBEF, rem, rfaHKYZ, rhaDRSCD, rimL, rmf, rna, rnb, rnhB, rnk, rpiBR, rpmHJ, rpsV, rspAB, sanA, sapBC, sdiA, sfa, sieB, slp, smf, smg, smpA, sms, sodC, sohA, soxRS, speBC, sprT, srlB, sspB, sugE, sulA, surE, syd, talC, tap, tdcCR, tdk, tehB, tesB, thiEH, thrLS, torADRT, tpr, treR, trkG, trpL, ubiX, ugpE, uhpT, uidA, umuD, ung, usg, uxaBC, vsr, wcaB, xapR, xasA, xerD and xylFH. Several operons have component genes that were apparently expressed and others that were not. Such inconsistencies (found in the dms, dpp, lac, nik, sap, and tdc operons) were not resolved. Genes without a known role are not listed.
The "well-defined, though quiescent" genes in cells growing exponentially in minimal medium were as follows: acpS, adiY, agaBDIV, alpA, aapZ, arsC, aslB, cadBC, caiBF, cchAB, cdsA, chpABRS, cmtAB, criR, csrA, cynST, deoR, ebgC, ecpD, emrB, envR, eutEJ, feoA, fimD_1, fimZ, fixAX, figAFH, frdC, frvARX, frwCD, fucAIKOU, galK, gcl, gefL, gip, glcG, glgS, glnBK, glpEGLTBK, glvBC, gntV, greA, grxA, gutM, hofB, hrsA, hyaE, hybFG, hycABFGHI, hydN, hypAC, kdgT, kdpC, hdtB, kefC, lacZYA, lar, leuO, malG, mbhA, mcrC, mhpB, mreD, nanA, nikBE, nirCD, nlp, nrdG, nrfBCFG, oraA, pheM, phnCDL, pinO, ppdABCD, priC, prmA, pshM, ptrB, racC, rfaZ, rhaD, rhsC, rnk, rpiR, rspB, sdaB, sieB, smpA, sms, sprT, srlABDR, tdcB, thrS, tnaB, ttdAB, ublH and uhpT. Genes of unknown function are not listed.
One hundred ten genes that were "silent" during transition of a culture from the exponential phase to the stationary phase in minimal medium are as follows: agaASW, ais, alkB, araFH, arsR, asr, bglG, ccmBCD, celA, cheWY, cpsG, cspBF, cvpA, cycW, dedA, dicBC, dsbE, dsrB, emrY, endA, evgS, fdnI, flgBCDGM, fliEFGHJLNOPQRT, gatR, glcD, gntKU, gusC, hdhA, hemK, hipB, hisMQ, hofFGH, holE, hrpA, hyaC, hybDE, hypD, kduI, lysR, malI, marBR, motA, napBCFH, narV, nuoAK, nupG, ogrK, pgsA, prfH, psrA, pspBC, pstA, pryL, rcsA, recT, relF, rhaR, rnb, sapBC, sdhD, sdiA, sfa, soxS, tdcCR, tdk, tpr, trkG, ubiX, uldAB, umuD, usg, and uxaB. Not listed are genes whose function is yet to be elucidated.
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ACKNOWLEDGMENTS |
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We thank T. Van Dyk, Z. Xue, L. Huang, and D. Smulski of the DuPont Company for helpful comments on this work. We are grateful to Dana Smulski for performing growth measurements. D. Zimmer and S. Kustu, University of California, suggested the mathematical formalism describing the data manipulations; we greatly appreciate that input.
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FOOTNOTES |
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* Corresponding author. Mailing address: DuPont Company, Central Research and Development, Biochemical Science and Engineering, Experimental Station, P.O. Box 80173, Wilmington, DE 19880-0173. Phone: (302) 695-9264. Fax: (302) 695-9183. E-mail: Robert.A.LaRossa{at}usa.dupont.com.
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REFERENCES |
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Top
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Introduction
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Discussion
Appendix
References
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Journal of Bacteriology, January 2001, p. 545-556, Vol. 183, No. 2
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.545-556.2001
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(2004). Patterns of large-scale genomic variation in virulent and avirulent Burkholderia species. Genome Res
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Buckles, E. L., Bahrani-Mougeot, F. K., Molina, A., Lockatell, C. V., Johnson, D. E., Drachenberg, C. B., Burland, V., Blattner, F. R., Donnenberg, M. S.
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Manna, D., Breier, A. M., Higgins, N. P.
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70: 2038-2043
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Anderton, J. M., Tokarz, R., Thill, C. D., Kuhlow, C. J., Brooks, C. S., Akins, D. R., Katona, L. I., Benach, J. L.
(2004). Whole-Genome DNA Array Analysis of the Response of Borrelia burgdorferi to a Bactericidal Monoclonal Antibody. Infect. Immun.
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Deng, S., Stein, R. A., Higgins, N. P.
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Mukhopadhyay, P., Zheng, M., Bedzyk, L. A., LaRossa, R. A., Storz, G.
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Moran, N. A., Plague, G. R., Sandstrom, J. P., Wilcox, J. L.
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Zhi, H., Wang, X., Cabrera, J. E., Johnson, R. C., Jin, D. J.
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Allen, T. E., Herrgard, M. J., Liu, M., Qiu, Y., Glasner, J. D., Blattner, F. R., Palsson, B. O.
(2003). Genome-Scale Analysis of the Uses of the Escherichia coli Genome: Model-Driven Analysis of Heterogeneous Data Sets. J. Bacteriol.
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Sabina, J., Dover, N., Templeton, L. J., Smulski, D. R., Soll, D., LaRossa, R. A.
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Han, M.-J., Jeong, K. J., Yoo, J.-S., Lee, S. Y.
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Marco, M. L., Legac, J., Lindow, S. E.
(2003). Conditional Survival as a Selection Strategy To Identify Plant-Inducible Genes of Pseudomonas syringae. Appl. Environ. Microbiol.
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Lia, G., Bensimon, D., Croquette, V., Allemand, J.-F., Dunlap, D., Lewis, D. E. A., Adhya, S., Finzi, L.
(2003). Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping. Proc. Natl. Acad. Sci. USA
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Choi, J. H., Lee, S. J., Lee, S. J., Lee, S. Y.
(2003). Enhanced Production of Insulin-Like Growth Factor I Fusion Protein in Escherichia coli by Coexpression of the Down-Regulated Genes Identified by Transcriptome Profiling. Appl. Environ. Microbiol.
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Dove, J. E., Yasukawa, K., Tinsley, C. R., Nassif, X.
(2003). Production of the signalling molecule, autoinducer-2, by Neisseria meningitidis: lack of evidence for a concerted transcriptional response. Microbiology
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Setty, Y., Mayo, A. E., Surette, M. G., Alon, U.
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Savli, H., Karadenizli, A., Kolayli, F., Gundes, S., Ozbek, U., Vahaboglu, H.
(2003). Expression stability of six housekeeping genes: a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J Med Microbiol
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Ojaimi, C., Brooks, C., Casjens, S., Rosa, P., Elias, A., Barbour, A., Jasinskas, A., Benach, J., Katona, L., Radolf, J., Caimano, M., Skare, J., Swingle, K., Akins, D., Schwartz, I.
(2003). Profiling of Temperature-Induced Changes in Borrelia burgdorferi Gene Expression by Using Whole Genome Arrays. Infect. Immun.
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Bina, J., Zhu, J., Dziejman, M., Faruque, S., Calderwood, S., Mekalanos, J.
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Xu, Q., Dziejman, M., Mekalanos, J. J.
(2003). Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro. Proc. Natl. Acad. Sci. USA
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Dennis, P., Edwards, E. A., Liss, S. N., Fulthorpe, R.
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Huang, L., McCluskey, M. P., Ni, H., LaRossa, R. A.
(2002). Global Gene Expression Profiles of the Cyanobacterium Synechocystis sp. Strain PCC 6803 in Response to Irradiation with UV-B and White Light. J. Bacteriol.
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Perriere, G., Thioulouse, J.
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Bernstein, J. A., Khodursky, A. B., Lin, P.-H., Lin-Chao, S., Cohen, S. N.
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Paustian, M. L., May, B. J., Kapur, V.
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Campbell, J. W., Cronan, J. E. Jr.
(2002). The Enigmatic Escherichia coli fadE Gene Is yafH. J. Bacteriol.
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Schneider, W. P., Ho, S. K., Christine, J., Yao, M., Marra, A., Hromockyj, A. E.
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Schut, G. J., Zhou, J., Adams, M. W. W.
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Helmann, J. D., Wu, M. F. W., Kobel, P. A., Gamo, F.-J., Wilson, M., Morshedi, M. M., Navre, M., Paddon, C.
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Lee, J.-M., Zhang, S., Saha, S., Santa Anna, S., Jiang, C., Perkins, J.
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Campbell, J. W., Cronan, J. E. Jr.
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DeLisa, M. P., Wu, C.-F., Wang, L., Valdes, J. J., Bentley, W. E.
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Zheng, M., Wang, X., Templeton, L. J., Smulski, D. R., LaRossa, R. A., Storz, G.
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Smulski, D. R., Huang, L. L., McCluskey, M. P., Reeve, M. J. G., Vollmer, A. C., Van Dyk, T. K., LaRossa, R. A.
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Van Dyk, T. K., Wei, Y., Hanafey, M. K., Dolan, M., Reeve, M. J. G., Rafalski, J. A., Rothman-Denes, L. B., LaRossa, R. A.
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