JOURNAL OF BACrERIOLOGY, Aug. 1993, p. 5145-5152 Vol. 175, No. 16 0021-9193/93/165145-08$02.00/0 Copyright C 1993, American Society for Microbiology Expression Analysis of Cloned Chromosomal Segments of Escherichia coli PUSHPAM SANKARt M. ELIZABETH HUTTON,t RUTH A. VANBOGELEN,§ ROBERT L. CLARK, AND FREDERICK C. NEIDHARDT* Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, 48109-0620 Received 11 January 1993/Accepted 4 June 1993 The novel transcription system of bacteriophage T7 was used to express Escherichia coli genes preferentially with a new low-copy-number plasmid vector, pFN476, to minimize toxic gene effects. Selected E. coli chromosomal fragments from an ordered genomic library (Y. Kohara, K. Ikiyama, and K. Isono, Cell 50:495-508, 1987) were recloned into this vector, and their genes were preferentially expressed in vivo utilizing its T7 promoter. The protein products were analyzed by two-dimensional gel electrophoresis. By using DNA sequence information, the gel migration was predicted for the protein products of open reading frames from these segments, and this information was used to identify gene products visualized as spots on two-dimensional gels. Even in the absence of DNA sequence information, this approach offers the opportunity to identify all gene products of E. coli and map their genes to within 10 kb on the E. coli genome; with sequence information, this approach can produce a definitive expression map of the E. coil genome. A central goal in biology is achieving a cellular paradigm, i.e., solving the structure of some particular cell and describ- ing how it functions. This intellectual feat will require the best efforts of reductionist approaches to learn the parts of a cell and how they function and the best efforts of systems analysis to discover how the parts are welded together into a functioning unit. There is little question that the bacterium Escherichia coli offers the best opportunity for achieving this paradigm. This is true, first, because of the vast amount of information gained from half a century of intensive study of this organ- ism. From mapping, sequencing, and identification of genes to investigation of gene products, metabolic pathways, gene regulation, and global regulatory networks, the wealth of our acquired knowledge of this organism far surpasses that of any other (11). The second advantage of E. coli is its unmatched accessibility by genetic, biochemical, and molec- ular techniques that facilitate both in vivo and in vitro analysis of its genome, its metabolism, and its higher-order functions of chemotaxis, stress reactions, genetic exchange, cell division, and molecular transport and translocation. Our laboratory has been committed to developing a cellu- lar protein data base for E. coli as a system of organizing and storing the vast amount of information about this cell. This data base is genomically linked, and the goal is ultimately to trace every protein to its structural gene and to account for every gene of the cell (23). Recent advances in DNA cloning and gene expression, in combination with two-dimensional (2D) gel electrophoresis to resolve the cell's complement of proteins, now make it feasible to create a complete linkage between a cellular protein data base and the genome of E. coli. In 1987 Kohara * Corresponding author. t Present address: Department of Microbiology, University of Texas Health Science Center, San Antonio, TX 78284-7758. t Present address: Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637. § Present address: Department of Biotechnology, Parke-Davis Research Division, Warner-Lambert Company, Ann Arbor, MI 48105. et al. (7) succeeded in constructing a physical map of the entire E. coli chromosome, indicating restriction sites for eight endonucleases along the entire 4,720 kb of DNA. As part of their project they created a minimal set of 476 hybrid lambda (X) phages, each containing a segment about 20 kb in length partially overlapping each of its neighbors. Preferen- tial expression of the genes from the members of this minimal set could conceivably allow the mapping to within 10 kb of every gene of E. coil. The RNA polymerase of bacteriophage T7 offers an obvi- ous means by which to achieve this preferential expression. A number of laboratories have utilized its property of selectively recognizing its natural promoters (13, 17, 18, 20). Various systems which use plasmid vectors carrying the T7 promoter in conjunction with the T7 gene 1 (coding for the RNA polymerase) located on a second plasmid (20), inte- grated into the E. coli chromosome (17, 18), or imported on a phage (17-19) have been designed. Our laboratory has created a new vector, pFN476, especially designed to ex- press all the genes on the ordered cloned fragments of Kohara et al. and to minimize the effects of toxic genes, thereby contributing to our goal of identifying all the gene products of E. coli. This vector has been used to analyze gene products from a dozen Kohara clones (24 pFNK plasmids, as each strand is cloned separately). The results from its use with three selected Kohara clones (six plasmids) are described here to demonstrate the extent to which plasmids are transcribed and translated and our methods of protein identification. MATERIALS AND METHODS Strains, phage, and plasmids. All strains listed in Table 1 are derivatives of E. coli K-12 except BL21(DE3) and PS1, derivatives of E. coli B (17, 18). The sources of plasmids and X phage clones are listed in Table 1. The minimal set of 476 hybrid phages containing overlapping segments that span the chromosome (7) was kindly supplied by K. Isono. Strain Q358 was used as the host for X clones. Initially, recombi- nant pFN476 plasmids were cloned in the commercially available strain DH5ao(F') to take advantage of its high 5145 on April 24, 2021 by guest http://jb.asm.org/ Downloaded from
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JOURNAL OF BACrERIOLOGY, Aug. 1993, p. 5145-5152 Vol. 175, No. 160021-9193/93/165145-08$02.00/0Copyright C 1993, American Society for Microbiology
Expression Analysis of Cloned Chromosomal Segments ofEscherichia coli
PUSHPAM SANKARt M. ELIZABETH HUTTON,t RUTH A. VANBOGELEN,§ ROBERT L. CLARK,AND FREDERICK C. NEIDHARDT*
Department ofMicrobiology and Immunology, University ofMichigan, Ann Arbor, Michigan, 48109-0620
Received 11 January 1993/Accepted 4 June 1993
The novel transcription system of bacteriophage T7 was used to express Escherichia coli genes preferentiallywith a new low-copy-number plasmid vector, pFN476, to minimize toxic gene effects. Selected E. colichromosomal fragments from an ordered genomic library (Y. Kohara, K. Ikiyama, and K. Isono, Cell50:495-508, 1987) were recloned into this vector, and their genes were preferentially expressed in vivo utilizingits T7 promoter. The protein products were analyzed by two-dimensional gel electrophoresis. By using DNAsequence information, the gel migration was predicted for the protein products of open reading frames fromthese segments, and this information was used to identify gene products visualized as spots on two-dimensionalgels. Even in the absence ofDNA sequence information, this approach offers the opportunity to identify all geneproducts ofE. coli and map their genes to within 10 kb on the E. coli genome; with sequence information, thisapproach can produce a definitive expression map of the E. coil genome.
A central goal in biology is achieving a cellular paradigm,i.e., solving the structure of some particular cell and describ-ing how it functions. This intellectual feat will require thebest efforts of reductionist approaches to learn the parts of acell and how they function and the best efforts of systemsanalysis to discover how the parts are welded together into afunctioning unit.There is little question that the bacterium Escherichia coli
offers the best opportunity for achieving this paradigm. Thisis true, first, because of the vast amount of informationgained from half a century of intensive study of this organ-ism. From mapping, sequencing, and identification of genesto investigation of gene products, metabolic pathways, generegulation, and global regulatory networks, the wealth of ouracquired knowledge of this organism far surpasses that ofany other (11). The second advantage of E. coli is itsunmatched accessibility by genetic, biochemical, and molec-ular techniques that facilitate both in vivo and in vitroanalysis of its genome, its metabolism, and its higher-orderfunctions of chemotaxis, stress reactions, genetic exchange,cell division, and molecular transport and translocation.Our laboratory has been committed to developing a cellu-
lar protein data base for E. coli as a system of organizing andstoring the vast amount of information about this cell. Thisdata base is genomically linked, and the goal is ultimately totrace every protein to its structural gene and to account forevery gene of the cell (23).Recent advances in DNA cloning and gene expression, in
combination with two-dimensional (2D) gel electrophoresisto resolve the cell's complement of proteins, now make itfeasible to create a complete linkage between a cellularprotein data base and the genome of E. coli. In 1987 Kohara
* Corresponding author.t Present address: Department of Microbiology, University of
Texas Health Science Center, San Antonio, TX 78284-7758.t Present address: Howard Hughes Medical Institute, University
of Chicago, Chicago, IL 60637.§ Present address: Department of Biotechnology, Parke-Davis
Research Division, Warner-Lambert Company, Ann Arbor, MI48105.
et al. (7) succeeded in constructing a physical map of theentire E. coli chromosome, indicating restriction sites foreight endonucleases along the entire 4,720 kb of DNA. Aspart of their project they created a minimal set of 476 hybridlambda (X) phages, each containing a segment about 20 kb inlength partially overlapping each of its neighbors. Preferen-tial expression of the genes from the members of thisminimal set could conceivably allow the mapping to within10 kb of every gene of E. coil.The RNA polymerase of bacteriophage T7 offers an obvi-
ous means by which to achieve this preferential expression.A number of laboratories have utilized its property ofselectively recognizing its natural promoters (13, 17, 18, 20).Various systems which use plasmid vectors carrying the T7promoter in conjunction with the T7 gene 1 (coding for theRNA polymerase) located on a second plasmid (20), inte-grated into the E. coli chromosome (17, 18), or imported ona phage (17-19) have been designed. Our laboratory hascreated a new vector, pFN476, especially designed to ex-press all the genes on the ordered cloned fragments ofKohara et al. and to minimize the effects of toxic genes,thereby contributing to our goal of identifying all the geneproducts of E. coli. This vector has been used to analyzegene products from a dozen Kohara clones (24 pFNKplasmids, as each strand is cloned separately). The resultsfrom its use with three selected Kohara clones (six plasmids)are described here to demonstrate the extent to whichplasmids are transcribed and translated and our methods ofprotein identification.
MATERIALS AND METHODS
Strains, phage, and plasmids. All strains listed in Table 1are derivatives of E. coli K-12 except BL21(DE3) and PS1,derivatives of E. coli B (17, 18). The sources of plasmids andX phage clones are listed in Table 1. The minimal set of 476hybrid phages containing overlapping segments that span thechromosome (7) was kindly supplied by K. Isono. StrainQ358 was used as the host for X clones. Initially, recombi-nant pFN476 plasmids were cloned in the commerciallyavailable strain DH5ao(F') to take advantage of its high
Strain, phage, or plasmid Relevant genotypea Source
StrainsQ358 hsdR supf +80r K. T. ShanmuganJM83 A(lac-proAB) +80dlacZAM15 gyrA' K. T. ShanmuganDH5a(F') F' hsdR AlacUl69 +80dlacZAM15 gyrA96 recAl Bethesda Research LaboratoriesBL21(DE3) F- hsdS lacLJ5::T7 gene 1 gyrA' F. W. StudierPSi BL21(DE3) srl-300::TnlO recAl This study
PhagesX clones 252, 253, 613 K. IsonomGP1-2 M13 with lacUV5::T7 gene 1 S. Tabor
PlasmidspGEM4 bla lacZ' MCS T7p PromegapKA101-6 pSC101 origin, bla par ArmstrongpFN476 psclOl origin, bla par lacZ' MCS T7p This studypFNK252L pFN476 + 252K insert, transcribed ccw This studypFNK252R pFN476 + 252K insert, transcribed cw This studypFNK253L pFN476 + 253K insert, transcribed ccw This studypFNK253R pFN476 + 253K insert, transcribed cw This studypFNK613L pFN476 + 613K insert, transcribed ccw This studypFNK613R pFN476 + 613K insert, transcribed cw This study
a MCS, multiple cloning site; 252K, 253K, and 613K, the insert from the Kohara recombinant X phage of the given number; cw, clockwise; ccw,counterclockwise.
cloning efficiency. This strain unfortunately carries gyrA,and pFN476 and similar pSC101-derived plasmids are notwell maintained (24). More recently we have employedJM105, another K-12 strain, already gyrA+, which we maderecA. The initial recombinants obtained in DH5a(F') wereexpressed shortly after construction or were transferred tostrain BL21(DE3), in which they are stable. This BL21(DE3)strain was also made to carry recA by Hfr mating.
Materials. Restriction endonucleases, T4 DNA poly-merase, Klenow fragment, calf alkaline phosphatase, 5-bro-mo-4-chloro-3-indolyl-3-D-galactopyranoside (X-Gal), andorganic chemicals were purchased from Boehringer-Mann-heim Biochemicals and Bethesda Research Laboratories.Ampicillin, NaCl, low-melting-point agarose, rifampin, andisopropyl-,3-D-thiogalactopyranoside (IPTG) were pur-chased from Sigma Chemical Co. Tryptone, yeast extract,and Bacto-Agar were supplied by Difco. DEAE-cellulose(preswollen) was purchased from Whatman.
Construction of pFN476. Plasmid pKA101-6 is a small (3.5kb), low-copy-number (one to five copies per cell) plasmidwith the pSC101 origin of replication and a unique BamHIsite in the nonessential part of the plasmid (2). This plasmidwas linearized at the BamHI site, and the ends were filled byusing Klenow fragment in the presence of deoxynucleosidetriphosphates. A 514-bp fragment from plasmid pGEM4(Promega technical bulletin no. 36), carrying a multiplecloning sequence, the LacZ a-peptide sequence (useful forcomplementing lacZAM15 to produce functional ,B-galacto-sidase), and the SP6 and T7 RNA polymerase promoters,was gel purified and blunt ended with T4 DNA polymerase.This fragment was ligated to the above-linearized pKA101-6and was used to transform strain DH5a(F'), picked, ana-lyzed, and transferred to strain JM83 for maintenance. Theresulting plasmid, pFN476, can be used as a vector intowhich E. coli fragments can be inserted for T7-dependentexpression (Fig. 1). It is small in size and low in copy numberso that large fragments (-20 kb), as well as genes whoseproducts can be toxic to the cell, can be cloned and stablymaintained. A multiple cloning sequence for easy manipula-tion and a blue or white screen for identifying recombinantplasmids is included.
Media and growth conditions. Strains for DNA preparationand cloning were grown aerobically at 37°C in Luria broth(10 g of tryptone per liter, 5 g of yeast extract per liter, and5 g of NaCl per liter) and when appropriate, 100 ,ug ofampicillin per ml. Media used for growth of strains forexpression of recombinant plasmids were morpholinepro-panesulfonic acid (MOPS) minimal medium with glucose (10)
pGEM4Z MCS EcoR 1 ...BamH 1...Hind 111
Pst 1
bla
FIG. 1. Cloning and expression plasmid pFN476. This plasmidcan be cut once in the multiple cloning site (MCS) with EcoRI,KpnI, SmaI, XbaI, AccI, SalI, and HindIII. There are two sites forAvaI, HincII, and PstI, with one of the sites for each in the MCS.There are three sites for BamHI, with one in the MCS.
E. COLI CLONED CHROMOSOMAL SEGMENT EXPRESSION ANALYSIS 5147
or glucose-rich MOPS (25), as indicated below in the sectionon expression. Cells containing recombinant pFN476 plas-mids were selected and screened on Luria broth agar plateswith 100 ,ug of ampicillin per ml, 0.5 mM IPTG, and 40 pg ofX-Gal per ml.DNA preparation. The procedure of Silhavy et al. (16) was
employed in the preparation of phage lysates. Phage X DNAwas prepared as described by Sankar et al. (15). PlasmidDNA was prepared by the method of Birnboim and Doly (3)as modified by Kreig and Melton (8a).
Isolation and purification of E. coli chromosomal DNAcloned in phage X. The purified recombinant phage X DNAwas digested with 1 U of EcoRI per pug of DNA for 7.5 minat 370C. The enzyme was then inactivated at 70'C for 10 min.With a 0.6% low-melting-point agarose gel containing 1 pg ofethidium bromide per ml, the DNA was separated by elec-trophoresis. The appropriate band (usually found betweenthe 20-kb left arm and the 9.6-kb right arm) was excised fromthe gel, and the DNA was recovered as described byManiatis et al. (9).
Construction of recombinant plasmids using pFN476. Plas-mid pFN476 was digested with EcoRI, and its 5' ends weredephosphorylated with calf intestinal alkaline phosphatase.The purified E. coli DNA fragments were ligated intopFN476, and the ligation mix was used to transform strainDH5a(F'). The transformed cells were plated on Luria brothplates with ampicillin, X-Gal, and IPTG (in amounts de-scribed above). White colonies were picked and screened forrecombinant plasmids. The size and orientation of the clonedfragments were calculated by standard procedures.
Transformation of strain PS1. In strain BL21, phage T7gene 1 coding for T7 RNA polymerase under the lacUV5promoter is integrated within the chromosome via phage XDE3 (17, 18). Strain PS1 is a recA derivative of strainBL21(DE3); it was transformed routinely by a modifiedCaCl2 procedure (16).
Expression of plasmid genes in strain PS1. Strain PS1bearing a recombinant pFN476 plasmid was inoculated inglucose-minimal MOPS (10) and shaken at 37°C overnightwith 50 ,g of ampicillin per ml. This culture was used toinoculate glucose-minimal MOPS (without ampicillin) andwas incubated at 37°C from an optical density at 420 nm(OD420) of 0.1 to 0.6. At this point IPTG was added to 0.5mM to induce T7 RNA polymerase gene 1, and the culturewas shaken for an additional 20 min. Rifampin was thenadded to 200 ,g/ml, and the culture was incubated withshaking for an additional 20 min. One milliliter of cells wasthen transferred to a prewarmed vial containing 25 ,Ci of[35S]methionine. After 10 min of labeling, the cells werechilled on ice, pelleted, and washed. Extracts for 2D gelanalysis were prepared as described elsewhere (22).
Expression of plasmid genes in strain DH5a(F') infectedwith phage mGP1-2. The expression of genes using phagemGP1-2 was adapted from the procedure of S. Tabor (19).Strain DHSa(F') bearing a recombinant plasmid was grownovernight in glucose-limited (0.04%) rich MOPS (25) (lackingmethionine and cysteine) with 50 pRg of ampicillin per ml.This culture was diluted to an OD420 of 0.5 and grown to anOD420 of 1.5 in glucose-rich MOPS (lacking methionine andcysteine) by shaking slowly at 37°C. At an OD420 of 1.5,phage mGP1-2 was added at a multiplicity of infection of 10and IPTG was added to a final concentration of 1 mM. Thecultures were shaken for 30 min, and then rifampin (200,ug/ml) was added. After an additional 30 min of slowshaking, the cells were labeled for 5 min with 25 ,uCi of[35S]methionine. After labeling, the cells were treated as
described above, and extracts were prepared for 2D gelanalysis.2D gel analysis. 2D gels were prepared as described in
reference 12, with 106 cpm of preferentially labeled plasmidproteins per gel. These extracts were electrophoresed bothalone (Fig. 2B) and mixed with extract from steady-statelabeled W3110 wild-type K-12 (Fig. 2D). The vector alone(Fig. 2A) and the steady-state labeled W3110 alone (Fig. 2C)were run as controls. Fluorograms were prepared and ana-lyzed for protein products of chromosomal inserts in therecombinant pFN476. DNA sequence information fromGenBank was used to deduce the molecular weight (MW)and amino acid composition of protein products by using thesoftware package of the Genetics Computer Group of theUniversity of Wisconsin. The amino acid composition wasused to calculate the pI of these proteins with the followingpKa for each of the residues: Asp, 4.5; Glu, 4.5; Cys, 9.0;Tyr, 10.0; His, 6.4; Lys, 10.2; Arg, 12.0; C terminus, 3.1;and N terminus, 8.0 (21). Some of these pKas differ signifi-cantly from other published pKas and may lead to calculatedpI values for proteins significantly different from those thatwould be obtained with some software packages, such asthat of the Genetics Computer Group.The apparent MWs and pIs of unidentified proteins ex-
pressed from plasmids were estimated from migration ofspots on gels, such as those shown in Fig. 2, in the followingway. (i) The vector spots (Fig. 2A) were identified anddiscounted. (ii) The spots expressed from the plasmid (Fig.2B) were identified on the gel containing the mixture ofplasmid and wild-type protein (Fig. 2D) and on Fig. C ifexpressed from the chromosome under our conditions. (iii)The location of an expressed spot in the pattern of wild-typespots (Fig. 2D) was determined and compared with a stan-dard reference gel (23) to assign standard reference gelx andy coordinates. (iv) The standard reference gel coordinateswere interpreted as the MW and pI on the basis of fittedequations relating the calculated MW and pI of a set of 148sequenced E. coli proteins with known standard referencegel coordinates (23). (v) Proteins are identified by matchingthe sequence-based MW and pI for a particular Koharainsert with the gel-based MW and pI of proteins expressedfrom the same Kohara insert.Some potential problems in spot analysis. Several potential
problems in spot analysis are anticipated. Severe streakingof a spot makes it impossible to assign a coordinate in thedimension of the streak, usually the pI dimension. A fractionof a percentage of spots are streaked under all protein loadlevels and film exposures, but nearly all others can beresolved by reducing the protein load or film exposure. Thestreaking in Fig. 4 is due to load and exposure levels used tobring up the faint spots on the same gel and resolves on alighter exposure.Due to the unusual conditions of transcription and trans-
lation there may be some aberrant or failed translation. Wehave observed great variation in the intensity of proteinspots on gels which may be due to differences of translationrate or possibly translational repression. In the 22 plasmidswe have described to date (this study and reference 23) weare not aware of any failed translation, although some spotsare certainly very light. In the very highly transcribed andtranslated proteins, aberrant proteins which differ slightlyfrom the normal protein in one or both dimensions are verycommon and show up as satellite spots. When the coordi-nates were previously known on the reference gel, the mainspot has invariably been the normal protein.
Spurious translation from internal start sites within a
FIG. 2. Expression of protein products on plasmid pFNK613L. Fluorograms from equilibrium 2D gels showing the proteins preferentiallylabeled with tritiated amino acids by the T7 expression method. (A) Expression from the vector pFNK476 alone. The three circled proteinsare Rep-101, 1-lactamase, and TnpR*, in descending order by size. (B) Expression from the recombinant plasmid pFNK613L. The spotscontributed by the vector are in small circles, and those from the 613 insert are in large circles. See Table 3 for protein identifications. (C)Expression of steady-state cell protein from the chromosome of W3110, wild-type E. coli K-12. The locations of the vector and insert spotsare indicated by small and large circles, respectively. Spots are actually present on this gel only if they are expressed from the chromosomeunder our steady-state conditions. (D) A mixture of the proteins expressed from pFNK613L and from the W3110 chromosome used to locatethe insert spots within the steady state spot pattern. Approximate scales for MW (in thousands on they axis) and pI have been provided forpanel D. See Materials and Methods for a fuller discussion of methods.
E. COLI CLONED CHROMOSOMAL SEGMENT EXPRESSION ANALYSIS 5149
single open reading frame is another potential problem whichcould lead to multiple spots scattered across the gel. We mayhave observed such a spurious translation in the extra spotobserved from the completely sequenced pFNK613L. If thisoccurs, it is certainly rare and the spurious spots can beremoved from the data base when the E. coli sequence iscompleted, since they will not be matched with any openreading frame.
RESULTS
Construction of recombinant plasmids of pFN476. The E.coli DNA fragments from clones 252 and 253 (mapped to min28), and 613 (mapped to min 76) of the Kohara minimal set(7) were each inserted in vector pFN476 in both orientations.These fragments were chosen for the first expression analy-sis because one area of the chromosome (covered by clones252 and 253) carries a high concentration of genes whoseproducts are identified on our reference E. coli 2D gels andthe other region has been completely sequenced (1, 4, 5).Because isolation of the E. coli DNA segments from the Xphage could be accomplished only by using incompletedigestion, restriction analysis was used to verify the size ofthe recombinant pFN476. In addition, care was taken toensure that each orientation of the insert was produced sopreferential expression from the T7 promoter would beperformed from both strands of DNA. The resulting plas-mids were named (Table 1) according to their originalKohara number and orientation in relation to the T7 pro-moter (e.g., pFNK613L is plasmid pFN476 containing E.coli DNA from Kohara clone 613 inserted so as to betranscribed counterclockwise [L] on the chromosome).Two methods of T7-dependent gene expression of recombi-
nant plasmids. Two similar methods of selectively express-ing genes using an inducible T7 RNA polymerase and itspromoter were used in this study. Both systems rely on thepolymerase under the lac promoter control, induced by theaddition of IPTG, and in both rifampin is employed to shutdown E. coli polymerase. The first system requires that theplasmid be expressed in strain PS1, a recA derivative ofstrain BL21(DE3) which includes an integrated T7 gene I atthe X int site. Transformation efficiency is low in this strain,so it is necessary to transform the newly constructed plas-mids into a more efficient cloning strain, complete thecharacterization of the plasmids, and then transform theminto strain PS1 for expression. A low basal level of T7 RNApolymerase is made from the integrated T7 gene I evenwithout IPTG, which presents a potential problem in cases inwhich the protein products of the cloned genes are toxic tothe cell (18). Nevertheless, the plasmids are low copynumber to minimize this problem, and we have had noinstance in which pFN476 recombinants could not be trans-formed into PS1 (22 of 22 plasmids [23]).
Nevertheless, a second system is available to bypasstoxicity problems that might occur; it employs the T7 RNApolymerase gene I under the control of the lac promoter andcontained within phage M13mplO (19). This alternativeprocedure is advantageous because there is no need for asecond transformation and, importantly, the T7 RNA poly-merase is available to the promoter only upon infection. Inthis procedure it is important to promote infection by keep-ing the pili intact through slow shaking and by optimizing themultiplicity of infection for the strain.
Analysis of protein products produced by chromosomalfragments. Genes located on plasmids pFNK252L,pFNK252R, pFNK253L, and pFNK253R are shown in Fig.
1310 1315 1320 1325 1330 1335 1340
EjoRi253
oppABCDF tonlYcL4BCDER rpABCDEL bruRsohB topA
FIG. 3. Chromosomal region containing the Trp operon andneighboring genes. This figure shows the region between positions1310 and 1340 on the physical map of E. coli (14). The positions ofgenes identified on clones 252 and 253 of the Kohara miniset areshown. This region has not been completely sequenced. The direc-tion of transcription for each gene is indicated by the direction of thediagonal lines (diagonal lines going down to the right indicatetranscription to the right and vice versa). The EcoRI site on 253 wasthe endpoint in plasmids pFNK253L and pFNK253R.
3. They were preferentially expressed in strain PS1 utilizingthe T7 RNA polymerase gene integrated on the chromo-some. The protein products of the trp operon found on theseplasmids were previously identified on our reference 2D gel(23). When the proteins from preferentially expressed geneswere analyzed by 2D gel electrophoresis, the known 2D gelspots were easily recognized (Fig. 4). In addition, the totalMWs of all the spots from these plasmids approximated theircoding capacity (Table 2). Preparation of these plasmidsleads to truncated genes. For example, pFNK252, on whichtrpB is not complete, yields a 2D gel pattern with anabbreviated protein product noted as TrpB*. However, thecomplete gene will usually exist on the neighboring Koharaclone because of the substantial overlap of these fragments.The sum of the 476 fragments is approximately 1.6 genomelengths. The truncated proteins will be noted as such in thedata base when the DNA sequence is known.The DNA nucleotide sequence of the 12-gene chromo-
somal fragment cloned in plasmids pFNK613L andpFNK613R is completely known (1, 4, 5). The 2D gelanalysis confirms that all but 2 of the 12 genes are tran-scribed clockwise (strain W3110 has a large inversion in thisregion) (Fig. 2; Table 3). The translated DNA of sequencesfound in GenBank was used to estimate the MW and pI ofproteins. In addition, estimates ofMW and pI were made onthe basis of migration of spots on the gel as described aboveand in references 21 and 23. After comparison of these datamost of the spots were identified as products of particulargenes (Table 3). Some genes on this fragment code forproteins with similar MWs and pIs, precluding a definiteidentification without further analysis. One extra proteinspot which does not correspond to any known open readingframe on pFNK613 was expressed.
DISCUSSION
The results reported indicate that the version of T7 poly-merase expression system developed here possesses manydesirable features. An advantage over previously describedsystems is that the integrated T7 gene I gives reliable, highexpression while the low copy number of the pFN476recombinant plasmids minimizes possible toxic effects fromuninduced expression. The prime requirement is that theexpression of genes on the cloned fragments be completeand be independent of normal physiological controls (induc-tion and repression). This requirement appears to be satis-fied, on the basis of two lines of evidence. First, there isvirtually total representation of the coding capacity of thechromosomal fragments in the polypeptide spots detected
FIG. 4. Expression of protein products on plasmids~pFNK,252R, pFNK252L, pFNK253R, and pFNK253L. Autoradiograms from the 2Dgels showing the proteins preferentially labeled with [31S]methionine by the T7 expression method. The numbered protein spots are uniqueto the plasmid. All other spots are either proteins encoded by the vector (circled) or are proteins made from RNA transcripts from E. coli RNApolymerase (cell background). (A) pFNK252L, which produced 11 proteins: 1, 10 kDa; 2, 12 kDa; 3, 12 kDa; 4, 14 kDa; 5, 18 kDa; 6, 21 kDa;7, TrpA; 8, 26 kDa; 9, TrpB; 10, TrpC; and a 12-kDa protein seen on the nonequilibrium gel. (B) pFNK252R, which produced 13 proteins:1, 12 kDa; 2, 13 kDa; 3, 15 kDa; 4, 16 kDa; 5, 20 kDa; 6, 24 kDa; 7, 32 kDa; 8, 32 kDa; 9, 36 kDa; 10, 57 kDa; and 3 proteins (8, 11, and 40kDa) seen on the nonequilibrium gel. (C) pFNK253L, which produced eight proteins: 1, 10 kDa; 2, 10 kDa; 3, 23 kDa; 4, 40 kDa (truncatedTrpB); S and 6, TrpC; 7, TrpE; and 8, TrpD. (D) pFNK253R, which produced 11 proteins: 1, 10 kDa; 2, 14 kDa; 3, 16 kDa; 5, 20 kDa; 6, 25kDa; 7, 50 kDa; 8, 27 kDa; 9, 27 kDa; 10, 33 kDa; 11, 35 kDa; and a 20-kDa protein on the nonequilibrium gel. Scales for MW are providedto assist the reader in evaluating the extent to which proteins transcribed and translated from the plasmid inserts utilize the available codingspace. See also Table 2. The scales were not used to assign MW to expressed proteins. Instead, MW was assigned as indicated in Materialsand Methods.
(Tables 2 and 3). Second, many individual genes that wouldnot be expressed during normal growth in glucose-minimalmedium, such as the polypeptides of the two anaerobicformate hydrogenlyase operons, have been observed in ourexpression analysis (23).Though barriers to the transcription of genes can be
presumed to be bypassed in this procedure, instances oftranslational repression may still be encountered. If severe,such translational barriers would restrict the quantity ofproduct. We have observed considerable variation in theamount of product from different genes on the same frag-ment (Fig. 4). It is hoped that the large amount of mRNA
produced in this system would still permit detectable proteinspots. A direct test of the seriousness of this concern isplanned through the use of fragments bearing genes knownto be translationally repressed, such as some of the operonsencoding ribosomal proteins.A second desirable feature of this system is the extent to
which protein synthesis is exclusively from mRNA producedby the T7 polymerase; rifampin at the concentration em-ployed is quite effective in shutting down the indigenous cellpolymerase, leading to fluorograms that display proteinsencoded by the recombinant plasmid and very little to nobackground. In fact, for matching the spots to the reference
a Coding capacity needed was calculated by summing the molecular massesof the products and converting to kilobases needed (1 kb encodes 37,037 Daof protein). Plasmid pFNK252 is about 18.3 kb in length, and pFNK253 isabout 16.8 kb (about 1 kb of the original 253 insert was not contained inplasmids pFNK253L and pFNK253R because of an internal EcoRI site).
gel pattern, it is necessary to prepare a second gel from theexpression extract to which a small quantity of extract fromstrain W3110 grown and labeled under steady-state referenceconditions has been added to provide a background pattern.The size of recombinant fragment in the Kohara collection
is close to ideal for expression analysis. The 20-kb fragmentsare readily manipulated and incorporated into the expressionvector. Resolution and identification by 2D gel electrophore-sis of the approximately dozen-and-a half genes on eachfragment are easily achieved, with little problem of overlapor crowding. Smaller fragments would increase the totalnumber of analyses needed per unit of length of chromo-some, and much larger fragments could present problems incloning, stability, and image analysis.Approximately 40% of the Kohara fragments have no
more than one internal EcoRI restriction site; 60 have noneat all. For these, the transfer of the fragment to our expres-sion vector by the methods described here is straightfor-ward. For the remainder, cloning is a more difficult task andit will be necessary to modify our protocol. We will attemptthe remainder with the Achilles cleavage technique (8), inwhich the internal EcoRI sites are preferentially methylated
TABLE 3. Proteins encoded and expressed from plasmidspFNK613L and pFNK613R
Plasmid and MW according to: pI according to:gene Sequence Gel Sequence Gel
livL 18,927 10.79 >7ftsS 21,677 21,500 6.29 6.02a These genes were previously identified.b The identification of FtsX and LivM and of FtsE and LivH could not be
distinguished on the basis of their MW, pI, and migration on the 2D gel.C FtsY has been reported to migrate as a dimer (6).d This spot does not correspond to any open reading frame in pFNK613L.
to block cutting prior to digestion of the flanking EcoRI sitesand subcloning. It will be necessary to use this and othertechniques as well as other sources of inserts to finishcloning the chromosome for expression.
Finally, it is noteworthy that approximately 46% of the E.coli genome has been sequenced, providing our expressionproject with ample information for virtually unambiguouslinkage of many expressed proteins to open reading framesof the fragment being analyzed. Although we expect muchhelp from the sequencing work under way in (and materialsbeing prepared by) the laboratories of Fred Blattner andDonna Daniels at the University of Wisconsin and GeorgeChurch at Harvard, the E. coli genome project in Japan is ofspecial interest to us because it is being carried out with theKohara recombinant phages, simplifying our use of se-quence information as it becomes available. Needless to say,it is also fortunate that the E. coli gene-protein data basederived from 2D gels has employed the same strain of E. coli(W3110) as used for the Kohara cloning.The results reported here indicate that complete expres-
sion analysis of the Kohara minimal set of cloned chromo-somal segments of E. coli is feasible.
ACKNOWLEDGMENTSWe thank Katsumi Isono for providing us several years ago with
the complete library of Kohara recombinant phages, Stanley Taborfor phage mGP1-2 and for helpful guidance in the T7 expressionsystem, Karen Armstrong for plasmid pKA101-6, F. W. Studier forstrain BL21(DE3), K. T. Shanmugan for strains Q358 and JM 83,and Alexander Pertsemlidis for suggesting that calculation of proteinisoelectric points from amino acid composition would be useful inthis work and for writing the algorithm to do so.
This work was supported by grant DMB-8903787 from the Na-tional Science Foundation.
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