FEMS Microbiology Letters 50 (1988) 17-23 17 Published by Elsevier

FEM 03131

pCON4 and pCON5" improved plasmid vectors to study bacterial promoters

Victor de Lorenzo, Marta Herrero and J.B. Neilands

Department of Biochemistry, University of California, Berkeley, CA, U.S.A.

Received 2 December 1987 Accepted 5 December 1987

Key words: Escherichia coli; Gene fusion; DNA, single-stranded antisense; Saturation mutagenesis; Promoter mutant; Plasmid

1. SUMMARY

Plasmid vectors pCON5 and pCON4 were de- vised to perform, with a single construction, a number of assay to characterize promoters of Escherichia coli (and related species) which usually require different steps of subcloning, fragment purification and radioactive labeling. Any DNA fragment with promoter activity can be cloned in these plasmids to generate either a lacZ-gene fu- sion (in pCON5) or a lacZ-operon fusion (in pCON4). Random and defined mutations to alter the activity or the regulation of the fusion can be obtained by directed saturation mutagenesis of the cloned fragment or site-directed mutagenesis using the single-strand form of the construction. Se- quencing of the mutations and in vivo assessment of their effects are carried out without any sub- cloning step. Additional analyses such as de- termination of the transcription start site(s) and in vitro 'footprinting' of DNA-binding proteins are also possible with the use of 5'-32p-labelled primers. As an example, up-mutations at the pro-

Correspondence to: V. de Lorenzo, Dept. of Biochemistry, University of California, Berkeley, CA 94720, U.S.A.

moter region of an iucA'-'lacZ iron-regulated fu- sion were isolated and analyzed with this proce- dure.

2. INTRODUCTION

Our laboratory is mainly interested in the regu- latory features of the high-affinity iron transport systems in microorganisms (see [1,2] for reviews). A model in which to study such regulation is the iron-controlled promoter of the aerobactin operon of plasmid ColV-K30 in E. coli [3]. In the course of the genetic and biochemical study of this pro- moter, we realized that several of the standard procedures to characterize the behavior of regu- lated E. coli promoters could be dramatically simplified by constructing a plasmid vector which combines in a single construction different fea- tures already existing separately in several availa- ble vectors.

In general, the transcriptional activity of E. coli promoters can be followed by constructing 'lacZ or 'galK gene or operon fusions under the control of the studied promoter [4]. Regulatory studies always involve the generation of mutants in the promoter region with altered phenotypes which, at

0378-1097/88/$03.50 © 1988 Federation of European Microbiological Societies

18

some stage, must be sequenced. Interesting muta- tions often fail to have an easily scorable pheno- type. In addition, predetermined base changes are sometimes desirable. Detailed examination of reg- ulation also requires mapping of RNA transcripts [5] and in many cases, footprinting experiments with purified regulatory proteins [6]. The oper- ations just listed usually take many different steps of subcloning, fragment purification and radioac- tive labeling.

Here, we describe two plasmids, pCON5 and pCON4, which in a single construction enable collection of a substantial amount of data regard- ing the promoter fragment which they contain. These vectors combine the advantages of the pre- viously described pMLB 1034 for gene fusion constructions [7] with those of the pGC2 vector [8] used for saturation mutagenesis, oligonucleotide- directed mutagenesis and sequencing. In addition, the resulting constructions permit the mapping of transcripts and DNase I footprinting analysis of DNA ligands without any fragment purification or subcloning step.

3. MATERIALS AND METHODS

3.1. Strains, plasrnids and DNA techniques E. coli strain CCl18 [F-A(ara-leu)7697

araD139 AlacX74 galE galK phoA20 thi rpsE rpoB argE(Am) recA1] was the recipient of the plasmids used throughout this work [9]. E. coli strain JM103 [10] was used for production of single-stranded (ss) plasmids [11] by superinfection of the corre- sponding transformants with M13K07 phage (Pharmacia). Plasmids pDB37 [12] pMLB1034 [7] and pGC2 [8] have been described previously. Standard recombinant DNA techniques [13] were used to construct pCON4 and pCON5 vectors (described in the corresponding RESULTS section). Plasmids carrying mutations at the aerobactin promoter are also described in RESULTS. DNA sequencing was carried out with the dideoxy method on ss plasmids [11] using as primer a 19-met (5'-CTGGCACGCGCTGGACGCG-3') synthesized by Bruce Malcolm, of this Depart- ment.

3.2. Saturation rnutagenesis of the aerobactin pro- moter region

The 78 base pair (bp) ssDNA insert of pDB37, which carries the iron-regulated promoter of the aerobactin system [12] was subjected to saturation mutagenesis as described by Myers [8]. Nitrous acid, formic acid, hydrazine, KMnO 4 and di- methyl sulfate were used as mutagenic agents. Mutated fragments were pooled and ligated to the untreated vector and the mixture was used to transform competent E. coli CCl18 cells, which were then plated out on iron-supplemented (50 gM FeC13) MacConkey-ampicilin (Ap) or LB-Ap plus 5-bromo-4-chloro-3-indolyl-fl-D-galactoside (XGal) plates [7]. Red colonies in MacConkey and intense blue colonies in XGal plates were picked up for further analysis (see RESULTS).

3.3. fl-Galactosidase assays E. coli CCl18 cells harboring the iucA'-'lacZ

fusion plasmids were grown in LB medium plus 150 ~tg/ml ampicillin up to an absorbance at 600 nm of approx. 0.1, when they were divided and added with either 50/xM FeC13 or 200/xM of the iron chelator 2,2'-bipyridyl. Aliquots of the cul- tures were taken during the following 4 h, and fl-galactosidase synthesis was determined as de- scribed by Miller [14].

4. RESULTS AND DISCUSSION

4.1. Construction of pCON5 and pCON4 vectors The steps followed for obtaining pCON5 are

summarized in Fig. 1. An approx. 2.8-kb EcoRI-HincII fragment from pGC2 was purified and ligated to a 3.4 kb EcoRI-BalI fragment from pDB37 [12]. The latter plasmid carries a iucA'-'lacZ gene fusion constructed by inserting 78 bp of DNA containing the aerobactin promoter sequence in the Sinai site of pMLB1034 [7]. The EcoRI-BalI fragment derived from pDB37 was used in the cloning strategy to facilitate the screen- ing of the first intermediate construction, pCON3. Subsequently, a 2-kb EeoRI-SacI fragment of pCON3 was excised and substituted by a 1.9 kb EcoRI-SacI fragment from pMLB1034 to restore the original cloning sites of that vector [7]. The

resulting approx. 6.2-kb plasmid, called pCON5, has the following characteristics: (1) As in the case of the widely used pMLB1034 [7], it allows the construction of 'lacZ gene fusions by inserting fragments with promoter activity and some struct- ural open reading frame in either of the EcoRI, SrnaI or BamHI sites, provided that they are in frame with the 'lacZ structural gene. (2) In ad- dition to a pBR322 origin of replication, it carries an M13 origin from pCG2 [8]. This affords a single-stranded form of the construction by super- infection of cells with wild type M13 [11] or derivatives like M13 K07 (Pharmacia). The orien- tation of the M13 origin specifies that the ssDNA which is extruded is the antisense strand of the 'lacZ fusion. (3) Immediately upstream from the EcoRI site, there is an approx. 300-bp, high-melt- ing-point fragment from pCG2, the 'CG clamp' [8], the utility of which will be described below.

The design of pCON4 is essentially the same than that of pCON5, except that a functional lacZ structural gene plus a ribosome-binding site from a trp'-'lacZ fusion was reconstructed downstream from the BamHI cloning site. This was made by inserting the 1.1-kb EcoRI-ClaI fragment of pRZ5605 [15] into the corresponding sites of pCON3 (Fig. 1). The new vector permits the con- struction of lacZ operon fusions by inserting a fragment with promoter activity in any of the cloning sites of the plasmids, regardless of the reading frame.

4.2. AppBcations of pCON4 and pCON5 A general strategy to study a bacterial promo-

ter with these 2 plasmids can be posited as fol- lows. The first step is to isolate a restriction frag- ment (ideally 150-300 bp, but may be up to 600 bp) carrying the promoter and construct a lacZ fusion in one of the 2 plasmids, preferably in pCON5. The promoter activity may be known in advance or can be assessed by constructing the fusion since the promoterless pCON5 vector ren- ders white colonies on XGal medium [7]. The choice between pCON4 or pCON5 to construct the fusion will depend on the nature of the in- serted fragment and the availability of restriction sites. If a gene fusion in pCON5 is obtained, the EcoRI-BamHI fragment which contains the in-

19

sert can be transferred, if desired, to the corre- sponding sites in pCON4 to generate an operon fusion in some cases, having the two constructions may be of interest if it is desired to compare the regulation of the transcriptional fusion with that of the transcriptional/translational fusion.

At this point, a number of directed mutagenesis protocols can be used to alter the regulation of the gene fusion. The method of choice for these con- structions is the so-called 'saturation mutagenesis' [8]. This procedure takes advantage of the changes in the melting temperature of the targeted frag- ment when single-base mutations are produced. To facilitate the separation of the mutated se- quences, the target DNA is located close to a short (300 bp) high-melting-temperature GC clamp, which pCON4 and pCON5 carry just up- stream of the cloning sites. Details for this proce- dure can be found in [8]. Briefly, ssDNA from the construction is obtained by superinfection of an F-containing E. coli strain transformed with the plasmid derivative. The DNA is then subjected to different chemical treatments which, under proper conditions, produce a random collection of base substitutions throughout the DNA at a prede- termined frequency. The ssDNA is then purified and hybr id ized with a 19-mer (5 ' -CTG - G C A C G C G C T G G A C G C G - 3 ' ) which is comple- mentary to the sequence of the GC clamp closest to the EcoRI cloning site. The annealed DNA is then treated with AMV-reverse transcriptase [13] in the presence of the 4 dNTPs and subsequently digested with EcoRI +BamHI. The resulting fragment carrying the collection of altered promo- ters is then inserted in the corresponding sites of the untreated vector pCON5 or pCON4. A step of enrichment in mutants can be introduced if the GC-clamp-containing restriction fragments are electrophoresed through a denaturing gradient gel (urea + formamide) in which the fragments carry- ing mutations will migrate either slower or faster than those which are intact [8].

Some regulatory mutants with easily scorable phenotype can be isolated directly, such as promo- ter-up or promoter-down mutants. The particular effect of the mutation can be assessed in the same fusion and the base change(s) localized by se- quencing its derived ss-form annealed with the

20 AEROBACTIN PROMOTER

ECO Bam Sal Eco Hint Xho Cla

POL YLINKEFt Primer Binding $~ ~ Pst Cla CG damp

Hind ~ A~p~r Hm ~ ) Hinc M13 o,t ori ~acZ

~ . ~ PSt

-2.9 Kb ~ Bal ~ " Sac

eco l ~ I pDB37 I-6"3 Kb , .

1.9 Kb fro m trp'-'lacZ

I J Sac 1.1 Kb \ \ pMLB1034 from \ \

ESB

{~ori 'lacZ

Cla

ESB Primer Binding Site ..._ 1~-4.~

CG clamp \ N_Y / Hind

~Cla //'~A t~- " ~

\ \ / u 'o;i ~ '

Cla

rbs trp'-'lacZ

/ S a c

[pCON 5 1~6.2 Kb I pCON 4 I-6.5 Kb Fig. 1. Genealogy of plasmids pCON5 and pCON4. Parent plasmids of the new vectors were pCG2 [8I and pDB37 [12]. Arrows inside the circles indicate the direction of transcription of the different genes. Arrows on top of pDB37 and pCON3 symbolize the location and orientation of an insert carrying the aerobactin promoter [12]. See text for explanation.

same CG-19-mer . The comple te p r o m o t e r reg ion can be sequenced in a single gel if the inser t is not too long. In some cases, it m a y be des i rable to use a second p r imer fur ther d o w n s t r e a m if larger se- quences are to be analyzed.

4.3. Up-mutations at the aerobactin promoter The 78-bp E c o R I - B a m H I inser t of the gene-

fusion p lasmids pDB37 and p C O N 3 , which carr ies the p r o m o t e r and the i ron- regu la t ion signals of the ae robac t in ope ron [12] was subjec ted to sa tu ra t ion mutagenes is as descr ibed above. P lasmids car ry ing mutagen ized f ragments were t r ans fo rmed into compe ten t E. coli C C l 1 8 cells and ampic i l l in - re - s is tant colonies which were red in M a c C o n k e y agar with excess i ron or deep b lue in L B - X G a l - i r o n pla tes were p icked as po ten t i a l ly deregula ted mutants . U p to one dozen i n d e p e n d e n t m u t a n t s were further ana lyzed Base changes at the p r o m o - ter region were local ized by D N A sequencing and

21

the effect of the m u t a t i o n s in respect to the i ron regula t ion was assessed by measur ing fl-galac- tos idase synthesis in the presence or absence of i ron as descr ibed in METHODS. U p o n D N A se- quencing, all of t hem tu rned out to have the same changed base (Fig. 2) cons is t ing of a G to A subs t i tu t ion at the th i rd c o d o n of the s t ructura l gene of the fus ion ( p M S series) or a G to T change in the same p lace ( p M U series). In addi t ion , p M U 8 also showed a G to T change close to the Shine- D a l g a r n o sequence (Fig. 2). Al l mu ta n t s had simi- lar responses to i ron concen t ra t ion , consis t ing of an approx. 10-fold increase in the f l -ga lac tos idase values in b o t h represssed ( + F e ) and induced ( + b ipyr idy l ) cond i t ions in respect to those of the

wi ld - type p r o m o t e r (Fig. 2). The base change in the deregula ted mu tan t s

found is l oca t ed in a l inker sequence which origi- na l ly does no t appea r in ei ther the lacZ gene or the ae robac t in p r o m o t e r sequence. W e th ink that

- - 1 Sau3A/BsmHI

I

+ I Met linker-m i ~S.D-I ~ r--

- - 78 bp

EcoRI -~-~ -I0

h 3tbpJ { Fur-FeZ+l

5'-TTATTATTTTACTGTGTAGGAGCTGTTTGATTATGATC GGGGATCCC GTC-3'->'lacZ

iucA' wt (70•860)

T (pMU8) ~/ T,A (1088110500)

Fig. 2. Up-mutants at the aerobactin promoter region. A 78-bp EcoRl-BamHI insert in pDB37 [12] and pCON3 contains the iron-regulated promoter of the aerobactin system. Figure shows promoter elements important for regulation [3,12]: the - 35 and - 10 sequences for RNA polymerase binding and the Fur-Fe 2+ primary operator (31 bp) in front of the iucA'-'lacZ gene fusion [12]. Transcription start site + 1 [3], the ribosome binding site (SD sequence) and the first codon of the structural gene are indicated in the enlarged sequence at the bottom. Only 2 codons of the original iucA gene are present in the fusion. These are followed by a short linker and the lacZ sequence. Arrows below the sequence indicate base changes observed in pMU and pMS series mutant plasmids. In all cases (see RESULTS), a G to A or G to T change led to about 10-fold increase in both repressed and induced fl-galactosidase levels afforded by the corresponding fusions. Bracketed numbers indicate the actual represssed( + Fe)/induced(+ bipyridyl) ratio of fl-galactosidase expressed in Miller units [14] in the wild type promoter (wt) and in one of the mutants (pMS4), the rest of them being in the same range (T.A). The additional base change G to T in pMU8 is also indicated, although/3-galactosidase levels afforded in that case were similar to the rest of the mutants.

22

the change may afford a more efficient translation initiation than the wild type counterpart, but probably it does not affect transcription. It is surprising that no derepressed mutants (i.e., aris- ing red colonies in MacConkey-iron plates) were obtained at the operator region for an iron-depen- dent repressor [12], the Fur protein, which con- trois virtually all iron-dependent genes in E. coli [16]. This fact is currently interpreted as the failure of single-base changes at the Fur-operator to af- ford strong derepression of the system.

4.4. Additional uses of pCON4 and pCON5 The major advantage of pCON4 and pCON5

to study bacterial promoters is to avoid the sub- cloning steps often associated to their characteri- zation. For instance, we have used the ssDNA form of the constructions as template for oligonucleotide-directed mutagenesis ([17], not shown). The resulting mutants, as in the case of saturation mutagenesis (see above), can be se- quenced and their behavior assessed in the same construction.

Although we have not tested them directly, additional types of experiments can be imagined with these constructions to characterize cloned promoters. For example, a convenient alternative to S1 mapping [5] to determine the transcription start sites is to use the ssDNA form of the con- structions hybridized with the RNAs (isolated in vitro or in vivo from plasmid-harboring cells) as the template for the 5'-labeled CG-19-mer exten- sion by T4 DNA polymerase [18]. Since the RNAs of the 'lacZ fusion carry the corresponding com- plementary sequence, it is also possible to use some of the commercially available - 4 0 M13 primers labeled at their 5'- end to map transcrip- tion starts by primer extension experiments with reverse transcriptase [19]. Finally, footprinting as- says may be performed with the supercoiled form of the plasmid derivatives by using the 5'-labeled CG primer [20].

The vectors described here, together with ap- propriate host strains, should, in principle, be useful to study regulated promoters of E. coli and closely related bacteria. A possible drawback is readthrough transcription from vector promoters. This makes pCON4 vector, in which the lacZ

sequence is headed by a ribosome binding site (Fig. 1), have a basal level of/3-galactosidase high enough to yield blue colonies in LB-XGal medium. However, we have observed (not shown) that the basal readthrough transcription in pCON4 and pCON5 is no higher than in plasmids in wide current use, such as pMLB1034 [7].

ACKNOWLEDGEMENTS

Plasmid pGC2 was obtained from Dr. R.M. Myers via Dr. A. Bindereif (Harvard University). VDL was a postdoctoral fellow (1F05TW03577- 01-B1-5) of the John Fogarty NIH Center and MH was a Fellow of the Fulbr ight /MEC of Spain Program.

REFERENCES

[1] Neilands, J.B. (1982) Annu. Rev. Nutr. 1, 27-46. [2} Winkelmann, G., Van der Helm, D. and Neilands, J.B.

(1987) Iron Transport in Microbes, Plants and Animals, VCH Verlagsgesellschaft, Weinheim.

[3] Bindereif, A. and Neilands, J.B. (1985) J. Bacteriol. 162, 1039-1046.

[4] Silhavy, T.J. and Beckwith, J.R. (1985) Microbiol. Rev. 49, 398-418.

[5] Aiba, H., Adhya, S. and De Crombrugghe, B. (1981) J. Biol. Chem. 256, 11905-11910.

[6] Johnson, A.D., Meyer, B.L and Ptashne, M. (1979) Proc. Natl. Acad. Sci. USA 76, 5061-5065.

[7] Silhavy, T.J., Berman, M.L. and Enquist, L.W. (1984) Experiments with Gene Fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[8] Myers, R.M., Lerman, L.S. and Maniatis, T. (1985) Sci- ence 229, 242-247.

[9] Manoil, C. and Beckwith, J. (1985) Proc. Natl. Acad. Sci. USA 82, 8129-8133.

[10] Messing, J., Crea, R. and Seeburg, P.H. (1981) Nucl. Acids Res. 9, 309-321.

[11] Zagursky, R.J. and Berman, M,L. (1984) Gene 27, 183-191.

[12] De Lorenzo, V., Wee, S., Herrero, M. and Neilands, J.B. (1987) J. Bacteriol. 169, 2624-2630.

[13] Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[14] Miller, J,H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[15] Mandecki, W. and Reznikoff, W.S. (1982) Nucl. Acids Res. 10, 903-912.

[16] Hantke, K. (1981) Mol. Gen. Genet. 182, 288-292. [17] Kunkel, T.A. (1985) Proc. Natl. Acad. Sci. USA 82,

488-492. [18] Chien-Tsung, M.T. and Davidson, N. (1986) Gene 42,

21-29.

23

[191 Proudfoot, N.J., Shander, M.H.M., Manley, J.M., Gefter, M.L. and Maniatis, T. (1980) Science 209, 1329-1336.

[20] GraUa, J.D. (1985) Proc. Natl. Acad. Sci. USA 82, 3078-3081.