LOW TEMPERATURE-REGULATED PROMOTERS IN E. COLI

FIELD OF THF, TNVENTTON

This invention generally relates to bacterial DNA molecules capable of initiating transcription at low temperatures. The invention- further relates to the expression of heterologous proteins at low temperatures

BACKGROUND OF THF, TNVENTTON

Many eukaryotic and prokaryotic proteins are insoluble when over-expressed in bacteria. They accumulate in the cytoplasm in the form of inclusion- bodies (IBs) . The protein in these IBs can be released only by using strong chaotropic reagents such as urea and guanidium hydrochloride. This process renders that task of isolating a soluble, active protein difficult (see, e.g., Schein, BioTftohnolnαy. 2:1141-1147 (1989) and Marino, BioPharm, 2:18-33 (1989)) . Several recent reports have indicated the utility of low temperature culture conditions in improving intracellular solubility or secretion efficiency of heterologous proteins expressed in E. coli. In the case of human interferon cc2 and γ, most of the protein was active and soluble when cultures were grown at temperatures lower than 30°C, whereas protein from cultures grown at 37°C was insoluble. A similar temperature dependence on solubility has been reported for the P22 tail spike protein, diphtheria toxin, ricin A chain, basic fibroblast growth factor, pro-subtilisin, and

SUBSTITUTE SHEET

lipooxygenase L-2 (Schein, supra) . It is thought that proteins produced at normal growth temperatures for _L_ coli (i.e., 37°C) can sometimes be folded improperly resulting in a complete or partial loss of the desired property of the proteins (e.g., biological activity). Thus, it seems that a potential simple method of obtaining heterologous proteins in a soluble form is to use a lower growth temperature for expression. Unfortunately, some other commonly used promoters in £___ coli (e.g., i_____, trp- lambda pL) may show reduced efficiency at low temperature. For example, the lambda Pτ_-based systems are incompatible with low temperature expression as they trypically require temperature upshift for gene expression. In addition to the potentially detrimental effect of high temperature on protein folding, this temperature upshift also results in induction of the heat shock proteins, several of which are proteases. Some other commonly used promoters (e.g. tac) seem to show reduced efficiency at low temperature. Accordingly, an inducible system adapted for use at reduced temperatures would be desirable.

In E. coli, there are a number of naturally- occurring proteins that show an increased rate of synthesis upon a temperature shift from 37°C to 10-20°C (Jones et al., J Baπ βrinl. __Lϋ:2092-2095 (1987)).

Recently, the gene encoding one of these proteins, cspA (or F10.6), which maps at 79 min on the E. coli chromosome, has been cloned. Although, this gene is stringently controlled and expressed at a high level when induced at low temperature, it is not clear at what level cspA regulation occurs. It is known that expression of cspA is sensitive to small temperature shifts and transitory in nature (Goldstein et al., Pro Na l Ac.ari Sci _r £2:283-287 (1990)). There are 13 other known proteins whose synthesis is cold shock inducible; seven of them have been identified (Jones et al., supra) . These include the produces of nusA, and ___a____. of

the nus-inf operon (69 min) pnp of the S15 operon (69 min) £e_-__. (58 min) , and ar-eE.F (3 min) . More recently, the hns gene (27 min) encoding the 15.4kDa nucleoid protein H-NS was shown, also, to belong to the cold shock regulon of E. coli.

It is thus an object of the present invention to identify, isolate and characterize additional E. coli promoters that are more active at low temperatures than normal growth temperatures (i.e., 37°C) .

SUMMARY OF THE INVENTION

The present invention relates to an isolated DNA molecule comprising a regulatory element isolated from E. coli that maps to 12, 34 or 81 minutes ±0.5 minutes of the E. coli genetic map, which when transformed or transfected into an appropriate host cell forms an expression system capable of initiating transcription of a heterologous gene when operatively linked to the 5' end of said gene, wherein the transcription levels obtained at 15 -20°C are at least 2 fold higher than transcriptional levels obtained at 3 °C.

In another aspect, the DNA molecule of the present invention comprises a DNA sequence (ID NO: 1, 2, 3 or 4) and functional equivalents of such sequences wherein such sequences have: at least 90% homology with SEQ ID NO: 1, 2, 3 or 4, and when transformed or transfected into an appropriate host cell forms an expression system capable of initiating transcription of a heterologous gene when operatively linked to the 5' end of said gene, wherein the transcription levels obtained at 15 - 20°C are at least 2 fold higher than transcriptional levels obtained at 37°C.

In related aspects, the present invention is a recombinant DNA vector comprising the DNA molecule of the present invention.

≡ST

In further related aspects, the present invention is a host cell transformed or transfected with the recombinant DNA vector of the invention.

The present invention further relates to a method for enhancing expression of heterologous proteins in bacteria which comprises: transforming or transfecting a bacterial host cell with the DNA molecule of the present invention; and culturing the host cell at a temperature below the normal growth temperature of the host cell; wherein the expression level of the heterologous protein obtained is at ^* least two-fold higher at the below normal growth temperature than the expression obtained at the normal growth temperature of the host cell.

The present invention still further relates to a method for enhancing expression of heterologous proteins in bacteria which comprises: transforming or transfecting a bacterial host cell with the DNA molecule of the present invention; initially culturing the host cell at the normal growth temperature of the host cell; and lowering the cell culture temperature to below normal growth temperature; wherein the expression level of the heterologous protein obtained is at least two¬ fold higher at the below normal growth temperature than the expression obtained at the normal growth temperature of the host cell.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 represents a circular reference map of __L__ coli K-12. The large numbers refer to map position in minutes, relative to the thr locus.

Figure 2 illustrates a temperature downshift induction of trpA-lacZ fusion -RNA in selected WQs strains. A northern blot analysis of total RNA from various E. coli strains using an EcoRI lacZ fragment

(from pCB267) as a probe is shown. Cells were grown in LB medium at 37°C (lanes 1,2,3,6) or cold shocked at

.15°C for 3 hrs. (lanes 4,5). E. coli wild type (+) IPTG lane 1 and (-) IPTG lane 2. Cold shock promoter transductants WQ3, lanes 3 & 4 and WQ11, lanes 5 & 6.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to DNA molecules which are capable of initiating transcription of a heterologous gene at levels which are higher at cold temperatures than at the normal growth temperature of the host cell. More specifically, the present invention comprises DNA molecules isolated from bacterial DNA in which transcription is induced at low temperatures. Furthermore, the DNA molecules of the present invention can also be transcriptionally induced in the presence of certain antibiotics.

The DNA molecules of the present invention comprise a regulatory element which comprises a promoter induced by a decrease in temperature, and optionally contains other regulatory regions which may also affect transcription (e.g., operators, enhancers, etc.). In addition, the DNA molecules can be induced by antibiotics in a manner similar to that proposed for the cold-shock response (see, e.g., Neidhardt et al., Proc Na l Apart Sc_i _r £2:5589-5593 (1990)) in which the addition of certain antibiotics known to inhibit ribosome function can mimic either a heat-shock or cold- shock response in E. coli. For example, treatment of E. coli WQ11 (a low temperature promoter - β galactosidase fusion; described more fully in the Examples section) with streptomycin did not induce expression of the β galactosidase protein. However, treatment with either chloramphenicol or tetracycline resulted in an 8-fold induction of β galactosidase expression. The DNA molecule of the present invention is isolated from a bacterial cell. Such bacterial cells may include, but are not limited to Salmonella,

Bacillaπeae. Pnβumococus, Streptococcus, and E. coli. Preferably, the DNA molecule of the present invention are isolated from E. coli. Alternatively, given the present invention, the DNA molecules of the present invention may be synthesized chemically, or even cloned and propagated as bacterial plasmids or as part of a phage library.

The DNA molecules of the present invention were isolated through PI genetic transduction using the transposon Tn5-lac. Tn5-lac is a derivative of Tn5 which can be used to generate promoter fusions expressing β galactosidase (β gal) (see, Kroos et al.. Prop. Na l Acad St.!- £1:5816-5820 (1984)). The Tn5-_L____. insertions that resulted in low temperature regulated β gal expression were identified by selecting for antibiotic resistant mutants that were capable of growth on lactose minimal medium at 15°C, but which grew poorly at 37°C on this medium. It is noted that the DNA molecules of the present invention may be isolated by a variety of via genetic techniques, and is not limited to the method disclosed above, i.e., use of genetic transduction using the transposon Tn5-lac. Other methods are known on the art, see for example, Miller, "Experiments in Molecular Genetics", Cold Spring Harbor Laboratory, CSH, NY (1972) .

The DNA molecules may range in length from a hundred base pairs to over ten thousand base pairs (10 Kbp) . Preferably the isolated DNA molecules range in length from lKbp to 9Kbp. It may thus be desirable to delete portions of the DNA molecule which have no effect on the transcription levels observed at reduced temperatures. Such manipulations are clear to those skilled in the art. Preferably, the regions upstream from the promoters of the present invention are not deleted. Such upstream regions may range from ten to two thousand base pairs.

In addition, the DNA fragments of the present invention were mapped on the E- coli chromosome (see Examples) . Genetic mapping is well known in the art, see for example. Glass, "Gene Function E. coli and its heritable elements". University of California Press, Los Angeles, CA (1982) or, B.L. Wanner, J Mo.l Biol. 1__J :39- 58 (1986) or Kohara et al., £__JJ_, __J2:495-508 (1987). The E. coli genetic map is divided into minutes, with 100 minutes representing the time taken for the entire E. coli chromosome to pass from an Hfr donor cell line to a F- recipient cell line during bacterial conjugation. The DNA mapping of molecules of the present is ^' disclosed in Table 5.

In addition, the DNA molecules of the present invention may comprise a DNA sequence as illustrated in Example 7 and functional equivalents thereof. Such functional equivalents are DNA molecules that when transformed .into an appropriate host cell forms an expression system capable of initiating transcription of a heterologous gene when operatively linked to the 5' end of said gene, wherein the transcription levels obtained at 15 - 20°C are at least 2 fold higher than transcriptional levels obtained at 37°C. In addition, the functionally equivalent sequences have a high degree of homology (i.e., at least 75% ho ology) to the sequences disclosed in Example 7. Preferably the degree of homology is at least 80%, more preferably it is at least 90%.

The DNA molecule may be operatively linked to a gene encoding a heterologous protein. That is, in the same reading frame and immediately upstream of a gene encoding a heterologous protein of interest. The present invention is not limited to any particular protein. Preferably, it is a protein that is mostly insoluble or biologically inactive when produced at normal growth temperatures in E. coli (i.e., 37°C), for example, human interferon oc2 and γ, human ccl-antitrypsin,

P. falciparu circumsporozoite proteins, HIV-1 proteins (e,g, tat) diphtheria toxin, etc.

The DNA molecule may also be found in a recombinant DNA vector capable of transforming or transfecting an appropriate bacterial host cell. Such host cells include, but are not limited to Salmonella, Baoillaceae, Pnp-umocociis. S rep ococcus _r and E. coli. Preferably, the host cell is E. coli.

Thus the expression system is a bacterial host cell preferably cultured in a nutrient-rich media where the expression level obtained of a heterologous protein is expressed at least two-fold higher at the below normal growth temperature than expression obtained at the normal growth temperature of the host cell. The normal growth temperature of the host cell is the optimal temperature in which the host cell propagates. For example, in E. coli the optimal temperature for growth is 37°C. The range of temperatures in which E. coli can grow is approximately 10°C to about 50°C. For E. coli, raising the temperature above 40°C or lowering it below 15°C (approximately) results in progressively slower growth, until growth ceases, at the maximum temperature of growth of about 49°C, or the minimum of about 8°C. The term low or reduced temperature refers to the culture temperature which is below the normal (or optimum) growth temperature. Preferably it is 12 to 27° below the normal growth temperature, or 10° to 25°C in E. coli; more preferably it is 17 to 22° below the normal growth temperature, or 15° to 20°C in E. coli. That is, a host cell transformed or transfected with the DNA molecule of the present invention expresses higher levels of a heterologous protein at 15° to 20°C than at 37°C. The levels of enhanced expression can range from 2 to 40 fold depending on the particular DNA molecule used and the method of induction (see Examples section) . Thus the present invention is also a method to enhance

expression of heterologous proteins by culturing a host cell at below normal growth temperatures.

Induction can be achieved by a ^" rapid decrease in temperature from 37°C to 15° or 20°C. This is referred to as "cold-shock". When the desired degree of growth is attained (i.e., monitored spectrophotometrically or other appropriate methods known in the art) , the temperature is rapidly lowered to a temperature greater than 9°C and less than 26°C. Preferably the temperature is in the 15 to 20°C range. In general, lower temperatures do not sustain practical growth rates. The culture is then grown in the lower temperature range for an appropriate period of time for optimum production of the heterologous protein of interest. The length of protein induction can be determined by pulse-chase experiments, SDS-PAGE electrophoresis. Western assays (if an antibody is available which recognizes the heterologous protein of interest) or other methods known to those skilled in the art. Alternatively, the host cell transformed or transfected with the DNA molecule of the present invention may be cultured at a low temperature (i.e., 15° to 20°C) without the need for a "cold-shock" protocol of the cell culture.

The following Examples are illustrative of the present invention. They are not to be considered as limiting of the present invention. Restriction enzymes and other reagents were used substantially in accordance with the vendors ^• instructions.

EXAMPLES

Bacterial strains and growth conditions. E. coli strain MC4100 (F-,Δ{ϋχ___E-_La__}U169, __£____139, _3_._I.150, relAl,

£__i___5301, j_______, deoC. EJ_______25) or its j_______Δ56 derivative strain SE5000 were used throughout this study

(Stratagene Inc., La Jolla, CA, USA or Silhavy et al., "Experiments with gene fusions." Cold Spring Harbor

Laboratory, CSH, NY (1984).) . For most culture manipulations bacteria were grown overnight aerobically at 37°C in LB medium (Miller, "Experiments in Molecular Genetics", Cold Spring Harbor Laboratory, CSH, NY (1972) ) . When necessary, medium was supplemented with 50μg/ml kanamycin or ampicillin. In cold shock experiments, a 1% inoculum from a starter culture was made into M9 minimal medium containing 0.2% glucose (Miller, H. supra) without antibiotic addition. Growth was monitored by measuring absorbance at 600nm ( ^A 600nm) . Temperature shifts were performed by transferring a portion of the culture into a water bath shaker, kept in a 4°C cold room and set at the appropriate temperature (15°C or 20°C) . Culture samples were collected at 1 hour intervals. For continuous growth conditions, cultures were kept at the appropriate temperature after initial inoculation and mid-log was reached in 2-days.

Genetic techniques. Standard procedures were used for preparation of PI phage lysates and PI genetic transduction essentially as described by Silhavy et al., supra, and is incorporated by reference herein. Chromosomal _]_______ transcriptional fusions were isolated by infecting SE5000 with PI: :Tn5-lac (see, e.g., Kroos, et al.. Prop. Na l. Apart. Sci. USA. _=_£:5816-5820

(1984) ) . Agar plates were placed in a Precision 815 low temperature incubator and kanamycin resistant colonies capable of growth on lactose minimal medium at 15°C after 4-5 days were selected. Mutants were isolated from separate transposition events.

β-Galactosidase assay. Quantitative β-galactosidase assays were essentially performed as described by Miller (supra) and is incorporated by reference herein. Bacterial cells were lysed using SDS and chloroform, β- Gal activity (Miller Units) was calculated as follows : [ (A420 ~ 1.75 x A550) x 1000] / [t (min) x v (volume of culture used in assay) x A500- • ^T e parent strain of

cold shock promoter transductants E. coli SE5000 does not show β-gal activityateither 37°C or 15°C.

Hfr and physical mapping. The mutations resulting from Tn5-lao insertions were transferred from SE5000 to

MC4100 by PI transduction; the cold inducible phenotype in these transductants was confirmed and these strains were then used as recipients in the Hfr mapping experiments. Hfr strains with TnlO insertions were obtained from Dr. Barbara Bach ann (CGSC, Yale

University School of Medicine, New Haven, CT, USA) . Rapid Hfr-mapping experiments were done as described by B.L. Wanner J. Mo.1. Biol.. _L_Ll:39-58 (1986)) and incorporated by reference herein. Streptomycin (50 μg/ l) and tetracycline (20 μg/ml) resistant exconjugants were selected on LB medium. Colonies were toothpicked and scored for lactose phenotype loss on either Mac ^' Conkey lactose indicator medium or LB-Xgal medium supplemented with tetracycline (25 μg/ml) . Rapid physical mapping of cloned promoters was achieved using the miniset collection of Kohara recombinant lambda phage library (Kohara, et al., £___ L, __!_:495-508 (1987) and incorporated by reference herein) . The membranes carrying the miniset were purchased from Takara Biochemical, Inc. (Berkeley, CA) . Hybridization conditions were as suggested by the manufacturer.

Antibiotic - mediated β-gal induction. The protocol described by VanBogelen and Neidhardt was essentially followed in conducting the antibiotic-mediated induction experiments (VanBogelen, R.A., and F.C. Neidhardt, Proc. Na l . Apart. Sci. USA, £2:5589-5593 (1990)). Briefly, ______ poli strain WQ11 was grown at 37°C in 0.4% glucose-amino acids-MOPS medium. When the culture was in exponential growth, the antibiotic chloramphenicol, tetracycline, or streptomycin was added at various concentrations. Treatment of bacterial cells with antibiotics at low

concentration for 75 min mimics cold shock from 37 to 20°C or heat shock from 28 to 49°C, while treatment with antibiotics at high concentration for 45 min mimics cold shock from 37 to 10°C or heat shock from 28 to 49°C, as appropriate.

Nucleic acid manipulations. For various nucleic acid manipulation techniques, standard procedures, described by Sambrook _____ .al. ("Molecular Cloning: A Laboratory Manual," 2nd Ed, Cold Spring Harbor Laboratory, CSH, NY (1989)) or Ausubel _____ _al. ("Current Protocols in

Molecular Biology," Wiley Interscience, New York (1987) ) , were followed and incorporated by reference herein. The EcoRI fragment of the laoZ gene from pCB267 (see, Schneider, K., and C. F. Beck, Gene, _____.:37-48

(1986)) served as a probe for Southern and Northern blot analyses.- This probe does not hybridize to either DNA or RNA from strain MC4100.

Cloning and sequencing. ρHC79 cosmid (BRL,

Gaithersburg, MD) digested with EcoRI was ligated with chromosomal DNA obtained from E. coli WQ mutants digested with the same enzyme, followed by in vitro packaging using Gigapackll Gold extract (Stratagene, La Jolla, CA) . E. coli SE5000 was then phage infected and ampicillin resistant blue transformants were selected on LB-Xgal plates. Subclones were generated in pBR322 or the promoterless probe plasmid vector pQF50 (Farinha, M. A., and A. M. Kropinski, .T. Baptfiriol. 122:3496-3499 (1990) ) . Double stranded plasmid DNA sequencing was accomplished using the Sequenase kit version 2.0 (USB, Cleveland, OH) as previously described (Kraft et al., BioTechnq. ___:544-547 (1988)) . A 22-mer primer extending from the left end of Tn5 into the transcriptional fusion site was used to carry out the sequencing protocol.

EXAMPLE 1

Isolation and identification of cold-inducible Tn5-_______, insertions. Cold-inducible transcriptional fusions to β-gal were isolated in E. coli strain SE5000 utilizing PI: :Tn5-lac. Tn5-lac is a transposable promoter probe which, when inserted in the correct orientation downstream from a promoter, creates a transcriptional unit producing a polycistronic trpA-laoZYA m-RNA (see, e.g., Kroos et al., supra) ) . Stop codons in all three reading frames prevent translation in the wrong reading frame of trp-lac fragments. Thus, observed enhancement of β-gal expression can almost certainly be attributed to transcriptional rather than translational activity. Randomly generated Tn5-1_____ chromosomal insertions that resulted in low temperature-regulated β-gal expression were identified by selecting for kanamycin resistant mutants that were capable of growth on lactose minimal medium at 15°C, but which grow poorly at 37°C on this medium. Employing this system, several colonies were isolated at a frequency of 0.01% and confirmed by β-gal cold induction in liquid culture. A total of seven mutants were selected for further studies.

TABLE 1. Level of b-gal expression (Miller Units) in _____ poll WQ11 at various temperatures

Growth Temperature b-gal ni s^ Fold Inrtupt.ion

Exponential Phase.

37°C

37 to 20°C ^a 37 to 15°C ^a 20°C 15°C

Stationary Phase

37°C

37 to 20°C ^a 37 to 15°C ^a 20°C 15°C

^a Where temperature shifts are shown, β-gal assays were performed 4 hours after the shift as described above.

-° Values are an average of two independent experiments in duplicate. Similar treatment of the parent strain E. coli SE5000 yielded no measurable β-gal units.

EXAMPLE 2

Low temperature inducible β-gal expression in strain WQ11. One of the first isolates which showed low temperature inducible β-gal expression was designated WQ11. In this strain, a temperature shift from 37°C to either 15°C or 20°C resulted in induction of β-gal expression with either exponential or stationary phase cells (see Tables 1 and 2) . The magnitude of the induction levels upon temperature downshift varied from

8 to 24 fold depending upon temperature and growth conditions. This induction level was maximum between 2-4 hours after temperature downshift. In mid-log cultures, continuous growth at either 20°C or 15°C resulted in a 36 to 42-fold higher β-gal expression, respectively, than in cells grown at 37°C. If cultures were assayed in stationary phase, cells continuously grown at low temperature typically had expression levels similar to that of cells subjected to a temperature down shift, i.e., 15-17 fold induction. Generally, the best β-gal induction by transient or continuous exposure to low temperature was observed using cells grown exponentially.

TABLE 2. Cold induction of β-gal expression in E. coli Tn5-1_____ insertion strains.

' Time (h) Average

Mutant β-σal Units ⁵ Fold Induction* ³

WQ1 0 89 ± 51 1 3 255 ± 28 3

WQ3 0 43 ± 10 1 3 309 + 156 7

WQ5 0 125 ± 85 1 3 600 ± 185 5

WQ6 0 81 ± 10 1 3 431 ± 106 5

WQ11 0 7.8 ± 0.5 1 3 95 ± 9 12

^a β-gal values are an average of three independent experiments in duplicate.

b Fold induction are over values for cells growing exponentially at 37°C.

EXAMPLE 3

Low temperature inducible β-gal expression in other Tn5- _La__ insertion mutants. The other cold inducible Tn5-la__ insertion mutants identified were designated WQ1 through WQ6. β-Galactosidase expression studies on these strains are shown in Tables 2 and 3. Mutants WQ2 and WQ4 were not analyzed in these studies since they were found to contain multiple Tn5-1____. insertions. Upon a temperature downshift from 37°C to 15°C, β-gal induction ranged from 3 to 12 fold (Table 2) in the various mutants. Exponential phase cells obtained from continuous growth at 15°C displayed β-gal levels that were roughly 2 to 3- fold greater than those seen in a 37°C to 15°C shift down, whereas stationary phase cells showed levels roughly equivalent to those observed in temperature downshift (Table 3) , except in the case of WQ6 where the levels in exponential and stationary phase cells were similar. In all of the isolated Tn5-lao mutants β-gal was expressed at 37°C, albeit, at lesser levels than during cold shock. Growing these mutants at 42°C or heat shocking them from 37°C to 42°C did not induce β- gal expression, but resulted instead in a slightly decreased β-gal expression when compared to levels at 37°C.

TABLE 3. β-gal expression after extended growth of E. coli Tn5-lac insertion strains at 15°C

Mutant Average, β-σal (Miller Units)

Fold Fold

Exponential Induction ^a Stationary Induction*>

Fold induction are over values from cells in exponential (a) or stationary (b) phase.

EXAMPLE 4

Induction of β-gal in strain WQ11 by selected ribosomally active antibiotics. It is currently thought that ribosomes can act as sensors of heat or cold shock in E. coli, based on their observation that antibiotics which target the ribosomes can mimic either heat shock or cold shock by inducing the full complement of the respective stress proteins. Treatment of E. coli WQ11 with streptomycin, an antibiotic reported to induce a response similar to heat shock, failed to induce β-gal expression. On the other hand, treatment of WQ11 with either chloramphenicol or tetracycline, shown to mimic a cold shock response, resulted in a 4-fold induction of β-gal expression in this strain (see e.g., Table 4). This is 2 to 3-fold lower than the induction observed upon temperature downshift (Table 1) . Similar results were found with E. coli strain WQ3. These results further substantiate the classification of the putative

promoters identified by transposon mutagenesis as cold shock promoters.

TABLE 4. Effect of antibiotic addition on β-gal expression in WQ11 at 37°C

Antibiotic f Cone . ) ^a β-σa 1 f Mi Her Tin i s ) b Fo 1 rt Induction

None 9 .7 1

Chloramphenicol (10 μg/ml) 35.0 4

Tetracycline (10 μg/ml) . 34.4 4

Streptomycin (10 μg/ml) 6.8 1

Chloramphenicol (2.5 μg/ml) 33.2 4

Tetracycline (0.5 μg/ml) 34.6 4

Streptomycin (4 μg/ml) 9.8 1

^a Treatment with antibiotics at high concentration for 45 min. mimics cold shock from 37 to 10°C or heat shock from 28 to 49°C, while treatment with antibiotics at low concentration for 75 min. mimics cold shock from 37 to 20°C or heat shock from 28 to 49°C.

-° β-gal values are an average of two independent experiments in duplicate.

EXAMPLE 5

Southern and northern analyses. To verify that the kanamycin resistant transductants resulted from transposition of Tn5-lac into the E. coli chromosome and that the selected mutants did not harbor multiple Tn5- lac insertions, a southern blot analysis was carried out on all mutants. Isolated chromosomal DNA digested with

EcoRI and probed with a labelled lacZ fragment resulted in a single hybridizable band, since there is one EcoRI site in Tn5-1_3___. In two of the transductants, WQ2 and WQ4, several hybridizable fragments were observed, indicative of multiple Tn5-la___ insertions and, thus, these mutants were not further characterized. Among the other five transductants, no two transductants displayed the same size hybridizable fragment indicating that the Tn5-lac transposition was likely to have occurred in 5 different regions on the E. coli chromosome.

The higher levels of β-gal activity exhibited by the mutants upon temperature downshift to 15°C could be attributed to increased transcription of the Tn5-lac cassette or to enhanced translation of β-gal protein. In an effort to distinguish between these possibilities, we performed a northern blot analysis the results of which are shown' in Figure 2. In E. coli mutants WQ3 and WQll, an elevated level of a 4.0 kb message, the expected size for the trpA-lacZ fusion, was observed at 15°C as compared to 37°C (Fig. 2, lanes 3,4,5 and 6) . No signals on the blot were detected from total RNA isolated from a mutant with a Tn5-J___c. insertion which does not express β-gal (not shown) or a wild type E. coli without IPTG induction (lanes 1 and 2) .

EXAMPLE 6

Cold shock promoter mapping experiments. Prior to cloning and sequencing the cold shock promoter regions from the various mutants, a rapid Hfr mapping technique was used to narrow down the map location of these Tn5- lac insertions on the E. coli chromosome. To date, eight out of the 14 known cold inducible genes have been identified and mapped (see e.g.. Table 5) . Based on Hfr mating results, WQll and WQ6 mapped to a region on the E. coli chromosome of 28-44 min and 12 min,

respectively. Mutants WQ1 and WQ3 mapped to 75-90 min, while mutant WQ5 mapped to 43-61 min.

In an effort to further pinpoint the chromosomal location of the cold shock promoter regions, rapid physical mapping using the miniset collection of the Kohara recombinant lambda phage library was carried out (see, e.g, Kohara et al.. Cell 50:495-508 (1987)), the results are shown in Table 5.

TABLE 5, Chromosomal location of cold shock genes in _____

M tant / Gene

WQl WQ3 WQ5 WQ6 WQll

cspA hns nus-inf operon

S15 operon recA aceE,F

^a ND, not determined.

EXAMPLE 7

Cloning and nucleotide sequence analysis. In an effort to complete the characterization of the cold shock promoters, they were first cloned and two of them, WQ3 and WQll which exhibit the best cold inducibility, were subjected to nucleotide sequence determination. EcoRI digested chromosomal DNA obtained from all mutants was

ligated to EcoRI digested cosmid vector pHC79. After In vitro packaging, E. coli cells were phage infected and ampicillin resistant, blue transformants were selected on LB-Xgal plates; then representative clones were evaluated for cold inducible β-gal expression. For ]___. coli strains WQ3 and WQll the clones were named pHC79- WQ3 and pHC79-WQll. Subclones from these cosmid inserts were generated in .pBR322 with the designation ρWQ3e and pWQllf. pWQ3e and pWQllf were sequenced, the results are presented below: ^'

pWQllf Seq ID No:l

GGAGGTTGCA TTTTACTGTC ATAGGTTACA ACATAGGCTG TTTTGAGAAG CCAATAGCGG 60

GCTTTATGCG 70

INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 70 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: -35_signal

(B) LOCATION: 6..11

(ix) FEATURE:

(A) NAME/KEY: -10_signal

(B) LOCATION: 62..67

Seq ID No:2

CACTAGCCCA AGGCCTTTTT CTTAGCCCGC CCTAGGCCCG CGGAGGTTGC ATTTTACTGT 60

CATAGGTTAC AACATAGGCT GTTTTGAGAA GCCAATAGCG GGCTTTATGC GGATAGCACA 120

ACAAC 125

INFORMATION FOR SEQ ID N0:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 125 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE: (A) NAME/KEY: -35_signal

(B) LOCATION: 47..52

(ix) FEATURE:

(A) NAME/KEY: -10_signal (B) LOCATION: 103. .108

pWQ3e

Seq ID No : 3

GTCCATTGCA GTGATCGGGT TGGCAAATAG TTATTACCCA ATTGCGAAGA A 51

INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: -35_signal

(B) LOCATION : 6. . 11

(ix) FEATURE :

(A) NAME/KEY: -10_signal

(B) LOCATION: 31..36

Seq ID No:4

CCCCCCGCCG ACGCCCAGCC TTAAGGGGAC GCGTGAAGTA AGAATAGTCC ATTGCAGTGA 60

TCGGGTTGGC AAATAGTTAT TACCCAATTG CGAAGAAGGG GATCGACCTT GAGAAAGCCC 120

ATTTCGGCGA CCTATAGGGT GTGCTTGCCC AACCCGTCGT TGTGCAAAAC GACTTTCCAA 180

ATTAGCGTTT GACCGTTTA 199

INFORMATION FOR SEQ ID NO: :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 199 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: -35_signal

(B) LOCATION: 52..57

(ix) FEATURE:

(A) NAME/KEY: -10_signal

(B) LOCATION: 77..82

While the above descriptions and Examples fully describe the invention and the preferred embodiments thereof, it is understood that the invention is not limited to the particular disclosed embodiments. Thus the invention includes all embodiments within the scope of the following claims.