Gene. 114 (1992) 103-107 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

103

GENE 06472

A simple and efficient system for the construction of phoA gene fusions in Gram-negative bacteriat (Envelope proteins; alkaline phosphatase; fl-lactamase; transposon; plasmid)

A.M. Duch~ne*, J. Patte, C. Gutierrez and M. Chendler Molecular Genetics and Microbiology Unit (CNRS), Toulouse (France)

SUMMARY

We have developed a two-plasmid system for generating gene fusions between phoA and cloned genes encoding envelope proteins. The vector plasmid carries a temperature-sensitive replication system and can be rescued at high temperature by insertion of an ISl-based transposon carrying the ori region of pBR322 and a phoA gene lacking transcription and translation initiation signals. The vector plasmid also carries the transfer origin of the conjugative plasmid, F, permitting transfer into a suitable recipient strain. We have used this system in the analysis of the bla gene cloned from pBR322.

INTRODUCTION

Envelope proteins play a central role in the interaction of the bacterial cell with its environment. Studies on these proteins have been greatly facilitated by the development of genetic approaches based on the use of gene fusions with Escherichia coliphoA (Manoil and Beckwith, 1985). PhoA is a periplasmic enzyme, that is inactive within the cell but is activated after translocation through the bacterial membrane (Boyd et al., 1987a). Translocation is facilitated by a signal sequence located at the N-terminus of the protein (Michaelis et al., 1986) but can be promoted by fusing PhoA Correspondence to: Dr. M. Chandler, Molecular Genetics and Microbiology Unit (CNRS), 118 route de Narbonne, F31062, Toulouse cedex (France) Tel. (33-61)33-59-82; Fax (33-61)33-58-86; E-Mail: Chandler @ FRCICT81. * Dedicated to the memory of Dr. J.-P. Lecocq. * Present address: Unit6 de G6nie Microbiologique, Institut Pasteur, 25 Rue Dr. Roux, F75724 Paris Cedex 15 (France)Tel. (33-45)68-88-28. Abbreviations: Ap, ampiciilin; Bla, fl-lactamase; bla, Bla-encoding gene (ApR); bp, base pair(s); Cm, chloramphenicol; IRL or IRR, left or right inverted repeat, respectively; IS, insertion sequence(s); kb, kiiobase(s) or 1000 bp; Kin, kanamycin; MCS, multiple cloning site(s); nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ori, origin of DNA replication; PhoA, alkaline phosphatase; phoA, gene encoding PhoA; R, resistance/resistant; s sensitive/sensitivity; Sm, streptomycin; Tc, tetracycline; Tnl0-Tek; TnlO modified by insertion of a Km R determinant within the tetA gene; XP, 5-bromo-4-chioro-3-indolyl-phosphatase.

with signal sequences of other proteins. PhoA export, and therefore activation, can also be directed by fusing it with integral membrane proteins on condition that the fusion occurs within a periplasmic domain of the protein. This property can therefore be exploited to define internal, external and transmembrane domains of integral membrane proteins (Manoil et ai., 1990). Special genetic tools have been constructed which permit isolation of fusions between a phoA gene missing the signal sequence and various bacterial genes (Manoil and Beckwith, 1985; Boyd et al., 1987b; De Lorenzo et al., 1990). We have previously described a family of transposable elements (omegons, f~), based on the insertion sequence I S I, which function in a wide variety of Gramnegative bacteria (Fellay et al., 1989; Joseph-Liauzin et al., 1989). Omegons can be used to perform generalised mutagenesis of bacterial genomes, to introduce foreign genes, and to directly select and screen for indigenous promoters. The aim of the present study was to construct an additional system designed to generate phoA fusions and to demonstrate the utility of this system in analysing the bla (Ap a) gene of pBR322. EXPERIMENTAL AND DISCUSSION

(a) The phoAfusion probe The transposon (omegon) carried by plasmid pEJL427 (Joseph-Liauzin et al., 1989) is composed of two minimal

104

a H B,

Km"

oriV

\_.B

IRL

pAMT1

Xb

(9276bp)

Tc R

phoA or/T

IS/* B-

~

~

R

IRR R

Sc*/Sm*

I

GAAI"rcGG'rAAI"GAOYOOAAG'lrlrAI"rGATAGAGTGGGTACCI~

AS*lAb*

pAMT6 (4650bp)

I PII

S Xh

~

P

R

H~

GGATCCTCGAGATCTCTGCAGGGTACCGAATrCCAGCTGAAGCTT B BII Kp PII

I

Be*

P

pAMT6:: bla

R Sm B

extremities of IS/(25 bp of IRR and 28 bp of IRL)flanking a Km R gene and the ori region of pBR322. Transposition functions are provided by a disabled copy of ISI located outside the transposon. The origin of transfer of the promiscuous transmissible plasmid RP4 enabling efficient delivery of the plasmid to various hosts, and a T¢ R gene used to distinguish transposition of the omegon from omegonmediated replicon-fusion (cointegration) are also located outside the transposon. A truncated phoA gene carried on a 2.6-kb SmaI-Xbal fragment of pPHO7 (Gutierrez and Devedjian, 1989), was inserted between the unique Scal and Xbal sites within the transposable segment of pEJL427 (and abutting IRR) to generate pAMT1. Since the extremity of IS/carries stop codons in two frames, the construction was designed so that phoA translation (following insertional fusion with an external translation unit) occurs in the third frame. The structure of this plasmid, together with the nt sequence of the I S / e n d and the 5' end of the truncated phoA gene, are shown in Fig. l a.

(b) The target vector To generate phoA fusions in the most direct fashion, we chose to use a two-plasmid system in which insertion of the omegon (which carries the replication system of pBR322) results in rescue of a target plasmid carrying the gene under study. For these purposes, we have modified plasmid pVFI6 (kindly supplied by V. Fran~;ois), a derivative of the temperature-sensitive pSC 101 plasmid, pH S 1 (HashimotoGotoh and Sekiguchi, 1977), which is compatible with Fig. !. Structure of plasmids pAMTI, pAMT6 and pAMT6::hla. Ah, Ahall; As, Asull; B, BamHl; BII, Bglll; Be, Bell, H, Hindlll: Kp, Klml; P, Psti; Pll, Pvull; R, EcoRl; Sc, Seal; Sm, Sinai; Xb, Xbal; Xh, Xhol. Asterisks indicate restriction sites destroyed in the cloning. (a)Plasmid pAMTI was constructed by inserting a 2.6-kb SmaI-Xbal fragment containing the 5' truncated plwA gene (Gutierrez and Devedjian, 1989) between the Scai and Kbal sites of omegon Km on pEJL427 (JosephLiauzin et al., 1989), Isolation and manipulation of plasmid DNA was carried out using standard techniques (Sambrook et al., 1989). The resulting 5,6-kb Km R transposable element carried by pAMTI is flanked by minimal IS/ends, IRL and IRR. The positions ofthe Km k and Tc Rgenes are indicated by boxes as is the truncated phoA gene and the disabled IS/ (IS !*); oriV is the ori ofpBR322 and oriT the origin of transfer of plasmid RP4. The nt sequence of the IRR-phoA junction is shown below. Stop codons are underlined and the phoA reading frame is boxed. (b) Plasmid pAMT6 (4.65 kb) consists of: a 2.9-kb BamHI fragment (from pHOl; G amas et al., 1986) carrying a temperature-sensitive pSC101 replication system, ori (rep ts), (Hashimoto-Gotoh and Sekiguchi, 1977), a 0.42-kb BgiII-Asull fragment containing oriT of plasmid F (Thompson and Taylor, 1982; Willetts and Wilkins, 1984; Thompson et al., 1989), a 1.33-kb Ahall fragment containing the CmR gene from pBR325, and a synthetic linker carrying BamHI, Xhol, BglII, Pstl, Kpnl, EcoRI, PvuIl, and HindIII sites. The nt sequence of this linker is shown below the p!asmid. Plasmid pAMT6::bla was obtained by insertion of a 1.15-kb Bell-Barn HI fragment containing the bla gene from p HP45::Tek into the unique BarnHI site of pAMT6.

105 pBR322. The essential characteristics of the target plasmid (pAMT6) are shown in Fig. lb. They include: the C m s gene, allowing the use of Cm for selection of plasmid fusions; an MCS for insertion of the test gene; and the origin of transfer (oriT) of the conjugal plasmid F, to permit transfer from the primary E. coli host into a suitable E. coil recipient. The latter property enables the eventual elimination of clones which may be generated by insertion of the plasmid into the host chromosome.

(c) lnsertional mutagenesis To evaluate this system we have used it to analyse the bla gene of pBR322. A DNA fragment carrying the entire gene was isolated from pHP45::Tek, a pBR322 derivative (Prentki and Krisch, 1984) carrying a Tnl0-Tek transposon (Fran~;ois et ai., 1987) inserted very close to the end of bla. This 1150-bp fragment, flanked by the BamHI site of pHP45 and the unique Bcll site of ISIO (Way et al., 1984), was inserted into the unique BonHl site of pAMT6 to generate pAMT6::bla (Fig. lb). The mature p-lactamase is coded by a 789-bp sequence (Sutcliffe, 1978) which is the target for the phoA + fusions. In an initial experiment, pAMT6::bla and the omegon probe, pAMT1, were transformed at 30°C into the Hfr strain PKI91 (Lcw, 1987) by selecting for Cm s (pAMT6::bla) and Tc s (pAMT1). Overnight L broth cultures, grown at 30°C with selection, were diluted 20-fold into fresh medium without antibiotics and grown at 42°C for approx. 4 h. The cultures were then incubated for one h at 37°C without agitation and mixed with the recipient CLG302 (Sin s) at a donor/recipient ratio of 1:3. Following mating for 30 min the cultures were spread on L agar plates supplemented with Km (for maintenance of the omegon), Sm, and the chromogenic PhoA substrate, XP. Blue (phoA +) colonies were easily detected among the exconjugants. Surprisingly, the majority of these clones appeared unstable upon purification giving rise to heterogenous blue and white sectored colonies. Since the omegon carries the ori region of pBR322 but lacks the negative regulator top gene, it seemed possible that the resulting high copy number interfered with cell growth. In order to determine whether a reduction in the plasmid copy number stabilises the fusions, identical matings were performed using CLG192 as recipient. This recA- strain ~arries the pcnBmutation which has been shown to lower the copy number of pBR322 (Lopilato et ai., 1986). The use of this recipient resulted in blue exconjugants which remained stable on further purification. When plasmids from six of these clones were transformed into DHB9, the pcnB + parent of CLG192, the resulting transformants again became unstable. Thus, the high copy number of the fusion plasmids appears to be responsible for their instability even in a recA- background.

To confirm the nature of the phoA + exconjugants, 16 independent mutagenesis experiments were performed using the pcnB- strain, CLG192, as final recipient. Blue colonies arose in the population of exconjugants with an average frequency of about 0.4% (within a range of 0.07 and 0.7%). Between one and eleven single colonies exhibiting the PhoA + phenotype were purified from each experiment and screened for resistance or sensitivity to Ap. In all but two cases (from a single mating) the exconjugants were found to be Ap s suggesting that omegon insertion had occurred within the bla gene. Omegon insertion into the bla gene should yield a PhoA ÷ phenotype in only one orientation and in only one of the three reading frames. Thus, random insertion into the bla gene should produce approx. sixfold higher frequencies of PhoA- Ap s clones. Consistent with this, screening 45 of PhoA- (white) exconjugants from each often experiments for their Bla phenotype yielded an average value of 2.8% Ap s clones compared to the average value of 0.4% of Ap s PhoA + clones. Plasmid DNA prepared from a single PhoA + (blue) Ap s clone from each experiment was analysed by agarose gel electrophoresis. The plasmids exhibited a size consistent with a single omegon insertion (5.6kb) within the pAMT6::bla plasmid (5.8 kb). The points and orientation of insertion were conveniently and unambiguously determined by digestion with EcoRl. As expected, the insertion had occurred within the bla gene of all fifteen independently isolated plasmids in an orientation consistent with transcription of the phoA gene from the bla promoter. We estimate that the localisation of the insertions is to within 50 bp. They can be divided into four major classes. An example of each class is shown in Fig. 2. The distribution of these insertions is presented in Fig. 3 and shows that, although a significant fraction of insertions occurred in the A+T-rich 5' region of the bla gene (Sutcliffe, 1978) as expected from the known insertion al specificity of I SI (Meyer et al., 1980; Galas e~ al, I980; Zerbib et al., 1985), a considerable number also cccurred throughout the gene. We have yet to determine whether this insertional specificity will prove problematic in the analysis of genes with a high G+C content. This two-plasmid system represents a useful addition to the available genetic probes for the analysis of exported and membrane-localised proteins. It is rapid, simple to use, and provides insertions at high frequencies and could in principle be used for creating phoA fusions in Gram-negative organisms other than E. coll. The presence of a pBR322 ori within the transposon permits subsequent one-step cloning in E. coli of the DNA flanking the omegon insertion. Moreover, although we have not determined here the point of insertion at the nt level, the use of suitable oligos homologous to the omegon ends provides a simple means for direct sequence analysis. The introduction of a direct plas-

106

S

1

2

3

4

u

S 0

Z :n:

1 = 0

H

H p3

F~oRI

7O

30

Fig. 3. Distribution of omegon insertions in the bla gene of pAMT6::bla. The processed Bla protein (hatched bar) and its signal sequence (solid bar) are indicated together with the bla promoter, P3, and the upstream EcoRl site. The upper panel shows the distribution of 15phoA +Ap s omegon insertions (open bars). The lower panel shows the A+T distribution in the region.

quence shown in Fig. la. This work was partially supported by contract 7691 from E L F - S A N O F I and a N A T O travel grant to M.C. and to D.J. G a l a s . Fig. 2. Agarose gel electrophoresis of EcoRl-digested plasn,id DNA. Plasmid DNA from phoA ÷ Ap s exconjugants representing each class of insertion was isolated by a standard mini-preparation technique (Sambrook et ai., 1989), digested with EcoRl, and subjected to dcctrophoresis on a 0.87/0 agarosv gel (lanes I-4). Lanes S are size standards (l-kb ladder, BRL). mid transfer step provides the additional advantage that all exconjugants carry an omegon insertion. Although in the results presented here we have used the bla gene to illustrate the utility of this system, we have also shown that other genes, such as osmC (Gutierrez and Devedjian, 1991) and certain of the membrane-localised F-transfer genes themselves (Willets and Skurray, 1987), are readily amenable to analysis using this system ( A . M . D . , unpublished data).

ACKNOWLEDGEMENTS W e would like to t h a n k J. Louarn and J.M. L o u a r n for m a n y stimulating discussions and material contributions, a n d V. Franqois for the generous gift of p V F 1 6 and p H P 4 5 : : T e k . Our thanks also to E. Joseph-Liauzin and M. K a g h a d (S.E.B.R.) for determining the phoA junction se-

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mutations on the kinetics of alkaline phosphatase export to the periplasm in Escherichia coll. J, Bacteriol. 167 (1986) 160-167. Prentki, P. and Krisch, H.M.: In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29 (1984) 303-313. Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Sutcliffe, J.G.: Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Prec. Natl. Acad. Sci. USA 75 (1978) 3737-3741. Thompson, R. and Taylor, L.: Promoter mapping and DNA sequencing of the F plasmid transfer genes traM and traJ. Moi. Gen. Genet. 188 (1982) 513-518. Thompson, T.L., Centola, M.B. and Deonier, R.C.: Location of the rick at oriT of the F plasmid. J. Mol. Biol. 207 (1989) 505-512. Way, J.C., Davies, M.A., Morisato, D., Roberts, D.E. and Kleckner, N.: New TnlO derivatives for transposon mutagenesis and for construction oflacZ operon fusions by transposition. Gene 32 (1984) 369-379. Wiiletts, N. and Skurray, R,: Structure and function of the F factor and mechanism of conjugation, in: Neidhardt, F.C., Ingraham, J.L., Low, K.B., Magasanik, B., Schaechter, M. and Umbarger, H.E. (Eds.), Escherichia coli and Sabmmella O'phimurium Cellular and Molecular Biology. Am. See. Microbioi., Washington, DC, 1987, pp. 11101133. Wiiletts, N. and Wilkins, B.: Processing of plasmid DNA during bacterial conjugation. Microbiol. Rev. 48 (1984) 24-41. Zerbib, D., Gamas, P., Chandler, M., Prentki, P., Bass, S. and Galas, D.: Specificity of insertion of IS/. J. Mol. Biol. 185 (1985) 517-524.