Gene, 111 (1992)21-26 © 1992 Elsevier Science Publishers B.V. AH rights reserved. 0378-1 I19/92/$05.00
21
GENE 06299
TnblaM: a transposon for directly tagging bacterial genes encoding cell envelope and secreted proteins (Ampicillin; spectinomycin; protein fusions;//-lactamase; mutagenesis; TnphoA; membrane proteins; exported proteins; recombinant DNA)
M. Tadayyonand JennyK. Broome-Smith Microbial Genetics Group, School of Biological Sciences, University of Sussex, Falmer, Brighton BNI 9Q,G (U.K.) Received by P.A. Manning: 19 August 1991 Revised/Accepted: 11 November/13 November 1991 Received at publishers: 5 December 1991
SUMMARY
A transposon, TnblaM, designed for the direct selection of bacterial mutants with insertions in genes encoding cell envelope and secreted proteins, was constructed and subcloned into plasmid and bacteriophage ;. delivery vectors. TnblaM is a spectinomycin-resistantderivative of Tn5 with an unexpressed open reading frame encoding mature//-lactamase (BIaM) at its left end. Therefore, when it inserts into genes in the correct orientation and reading frame, gene fusions encoding hybrid proteins are generated. By introducing TnblaM into bacterial cells and selecting ampicillin-resistant (Ap R) colonies, the subset of isolates producing extracytoplasmic BIaM, and hence containing TnblaM inserted in genes encoding secreted proteins and cell envelope proteins, can be directly selected. TnblaM, fike TnphoA, can therefore be used to preferentially mutagenise genes encoding extracytoplasmic proteins, but is has the advantage over TnphoA that the desired mutants can be isolated by direct selection (as Ap R colonies) rather than by phenotypie screening. Isolates in which TnblaM occupies sites in the chromosome from which it can transpose at high frequency are readily identifiable, and constitute TnblaM donors, with which to simply and efficiently generate rare types of insertion mutants. Moreover, the Ap R selection that is used with TnblaM can be fine-tuned to obtain blaM fusions to poorly or well-expressed genes.
INTRODUCTION
Gene fusion studies have proved immensely valuable in the analysis of gene expression and protein structure and Correspondence to: Dr. J.K. Broome-Smith, Microbial Genetics Group, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG (U.K.) Tel. (44-0273)606755; Fax (44-0273)678433. Abbreviations: Ap, ampicillin;Bla,//-lactamase; BlaM, mature portion of Bla; blaM, 5'-truncated bin gene coding for BIaM; bp, base pair(s); IS, insertion sequence; kb, kilobase(s) or 1000bp; Km, kanamycin; MIC, minimum inhibitory concentration; nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide-gelelectrophoresis; R, resistance/resistant; s, sensitivity/sensitive;Sp, spectinomycin; Tc, tetracycline; TEM, specifies the source of Bla; Tn, transposon; XP, 5-bromo-4-chloro-3-indolyl phosphate; [ ], denotes plasmid-carrier state.
function. Two translational fusion systems have been developed specifically for analysing the organization of proteins within the bacterial cytoplasmic membrane and identifying bacterial export signals. In both systems hybrid proteins are generated by fusing the coding regions for reporters that display location-specific phenotypes to progressively truncated forms of the target genes. In the first system the mature form of alkaline phosphatase is used as the reporter. Only extracytoplasmic forms of alkaline phosphatase are enzymatically active. Therefore, by plating cells onto agar containing XP, a chromogenic substrate for alkaline phosphatase, all isolates in which alkaline phosphatase has been fused to translocated portions of proteins can be identified as blue-coloured colonies. Hence this reporter provides a way of screening for cytoplasmic memhrane export signals (Manoil and Beckwith, 1985). In the
22
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Fig. 1. Construction and structure of TnblaM. Only the relevant restriction sites are shown. (a) Construction of TnblaM. Unless otherwise stated, the suppressor=free strain E. coil HB2151 (Alac-pro, thi, [F', proA*B ÷, lacI°ZAMIS, traD36]) was used for transformations and was grown in L broth or on L agar (Miller, 1972) supplemented with 50/Jg Km/ml, 20 #g Sp/ml, 20 pg Tc/ml or Ap (variable concentrations) as required. A derivative of pBR322 with Tn5 inserted at nt 31-39 was provided by Dr. D.E. Berg, Washington University, St. Louis, MO. This plasmid was digested with BamHI and the fragment carrying ISSOR was inserted into the BamHI site of pBS~- (Spratt et al., 1956) to produce pJBS647. Plasmid pJBS647 DNA prepared from a dam- strain was linearised with Bcll, and ligated to the 2-kb BamHl fragment encoding Sp R (the ~ cassette) obtained from pHP45f~ (Prentki and Krisch, 1984) to produce pJBS648. Plasmid pJBS646 contains blaM inserted as a PruII-AhaIII fragment into the HincII site of pteHl9 (Spratt et al., 1986) and encodes a cytoplasmic form of BlaM (Broome~Smith and Spratt, 1986). This plasmid was linearised by partial AhaIII digestion and ligated to the end-filled BamHl fragment from pJBS648 containing the Sp R gene, to yield pJBS649 in which blaM precedes the Sp R gene and all of the ISSOR sequences that are essential for Tr6 transposition. Finally, pBRG700, provided by Dr. D.E. Berg, was used as a source of an Alul-BamHi fragment
23 second system the mature form of BIaM is used as the reporter. In this case translational fusions to both cytoplasmic and extracytoplasmie portions of target proteins can be readily identified since isolates producing BIaM fusion proteins can grow when plated at high inocula onto agar containing Ap. However, only translocated forms of BIaM are able to protect individual cells against lysis by Ap. Therefore, isolates in which the BIaM moiety is extracytoplasmic can be distinguished from those in which it is cytoplasmic by their ability to survive and form colonies when plated at high dilution onto agar containing Ap (Broome-Smith and Spratt, 1986). Moreover, by selecting Ap R colonies the subset of isolates in which BlaM is fused to extracytoplasmic domains of proteins can be directly isolated. Hence when BlaM is used as the reporter, cytoplasmic membrane export signals can be directly selected (e.g., see Zhang and Broome-Smith, 1989). Plasmid vectors for the construction of alkaline phosphatase fusions to cloned genes in vitro, and a transposon, TnphoA, for the insertion of the mature alkaline phosphatase coding region into plasmid and bacterial chromosomes in vivo, have been constructed and widely used to screen for genes containing export signals (Hoffman and Wright, 1985; Manoil and Beckwith, 1985; 1986; Manoil et al., 1989). Plasmid vectors for the generation of blaM fusions in vitro to cloned bacterial and eukaryotic genes expressed in E. colt have also been described and used to analyse membrane protein topology (Broome-Smith and Spratt, 1986; Zhang and Broome-Smith, 1990; reviewed in Broome-Smith et al., 1990). We report here the construction of the Tn5 derivative TnblaM and show how it can be introduced into Gram- bacteria and used to directly select mutants with TnblaM insertions in genes encoding cell envelope and secreted proteins.
RESULTS AND DISCUSSION
(a) Construction of TnblaM Tn5 is a composite Tn in which inverted repeats of the smaller transposable elements IS.S0L and ISSOR bracket a central region that encodes Km R (Berg and Berg, 1983). Genetic analysis has revealed that only the transposase gene of ISSOR and the outermost 19 bp of each IS are required for the efficient transposition of any intervening sequences (Rothstein et al., 1980; Sasakawa et al., 1983; Johnson and Reznikoff, 1983). Moreover, Tn5 and its derivatives are able to transpose in a broad range of Grambacteria and display a relatively low level of insertional specificity, making them ideal elements with which to mutagenise the genomes of many different bacteria (reviewed in De Bruijn and Lupski, 1984). The strategy used to make TnblaM was to fuse the outer terminus of ISSOL to the start of blaM, such that the ORF encoding BIaM was extended back to the outer end of ISSOL, and to insert a S p R determinant between the modified BlaM-coding region, and the transposase gene and outer terminus oflSSOR. The construction ofpJBS650 that carries TnblaM, and the relationship of TnblaM to TnS, are outlined in Fig. 1. (b) Delivery of TnblaM into the Escherichia coli chromosome A plasmid vector for delivering TnblaM into the E. colt chromosome was made by subcloning the transposon from pJBS650 into a pSC101 derivative that is temperaturesensitive for replication in E. colt (Fig. 2a). DNA of the resultant plasmid, pJBS651, was used to transform the strain JMI01F- (Aiac-pro, tht, supE) to Tc R at 30°C, and a transformant was purified at 30°C on L agar containing
containing the outer 19 bp of ISSOL (Sasakawa et al., 1983) and this fragment was inserted between the Smal and BamHI sites of pJBS649, yielding pJBS650 in which the blaM ORF is N-terminally extended, back through the outer terminus of IS50L. Therefore, pJBS650 contains a novel Tn, TnblaM, that possesses all of the sequences necessary for the efficient transposition of the Spa marker and the modified BlaM-coding region. IS sequences are boxed. (b) The relationship of TnblaM to TnS. The stippled and wavy shading denote the BlaM-coding region and fl cassette, respectively. The sequence from the left end of TnblaM to the third codon of the BlaM coding region is: 5'-CTG ACT CTT ATA CAC AAG TCG GGA TCC GTC CTG CGT CAC CCA GAA... Leu Thr Leu Ile His Lys Ser Gly Ser Val Leu Arg His Pro Glu... +1 +2 +3 Abbreviations: A, Ahalll; B, BamHl; Be, BclI; E, EcoRI; EF, end-filled; H, Hindlll; lacpo, lac promoter and operator; P, Pstl; RV, EcoRV; S, Sail; Sin, Sinai; Sph, Sphl; StrR, streptomycin-resistance marker. Symbols × indicate cleavage by restriction enzymes. Fig~ 2. Plasmid and bacteriophage vectors carrying TnblaM. Only the relevant restriction sites are shown. (a) Construction ofpJBS651. Plasmid pPM 121, a pSCI01 derivative that is temperature-sensitive for replication in E. colt (Meacock and Cohen, 1979)was obtained from Dr. P.A. Meacock, University of Leicester, U.K. TnblaM was cloned into pPMl21 by digesting pJBS650 with EcoRI + EcoRV, and ligating the digested DNA to pPM 121 DNA that had been cut with EcoRI + Smal, yielding pJBS651. (b) Construction of ,tTnblaM. Plasmid pJBS650 DNA was digested with EcoRV and an EcoRI linker was added by linker tailing (Lathe et al., 1984) to produce pJBS652. Phage ANM627 has the genotype A(srlA 1.2) c1857 srl,[4 ° nin5 srl,~5 ° Sam7 and was obtained from Dr. N.E. Murray, Department of Molecular Biology, University of Edinburgh, U.K. DNA of ANM627 was digested with EcoRl, and the left and right arms of the bacteriophage were purified and ligated to pJBS652 DNA that had been digested with EcoRl, to produce ,[TnblaM. Abbreviations as in Fig. 1 legend.
24 20 pg Tc/ml and 20 #g Sp/ml. To obtain E. coil derivatives carrying TnblaM in the chromosome, JMI01F- [pJBS651 ] was then grown for approx. 20 generations at 42°C in the absence of any antibiotics (to dilute out the plasmid), and Sp R isolates were selected. Less than 1% of these were Tc R, and examination of the DNA content of cell lysates (Holmes and Quigley, 1981) revealed that they did not contain any pJBS651 plasmid DNA (data not shown). Most (approx. 70%) of the above isolates failed to form an area of complete growth when inoculated at high cell density (by toothpicking colonies) onto agar containing 10 #g Ap/ml, suggesting that TnblaM had inserted into the chromosome such that its BIaM coding region was unexpressed. However, the remaining isolates formed an area of complete growth in this 'patch test', indicating that blaM was either fused in-frame to an N-terminal portion of a chromosomal gene, or that it was fused out-of-frame to aa N-terminal portion of a relatively well-expressed gene (diEcussed in Broome-Smith and Spratt, 1986). Isolates in which transposition of TnblaM into the chromosome had resulted in the fusion of BIaM to extracytoplasmic portions of E. coil gene products were directly selected by plating aliquots of the culture of JM101F-[pJBS651] that had been grown in antibioticfree medium at 42°C (see above) onto L agar containing Ap. When Ap was used at a concentration of 10 #g/ml (i.e., at twice the concentration that lyses non-fl-lactamase producing cells of E. coil or cells producing cytoplasmic forms of//-lactamase), 1 in 4 × 105 cells grew and formed colonies. Several of these isolates were purified and then plated at high dilution onto agar containing 5 ~tg Ap/ml. Individual cells of each isolate were able to grow and form colonies under these conditions, confirming that they produced extracytoplasmic forms of Bin. Hence, by combining a method for delivering TnblaM into the E. coil chromosome with selection for Ap R colonies, mutants of E. coli with TnblaM inserted in genes encoding extracytoplasmic proteins were directly selected.
(e) Identification of strains that display efficient TnblaM transposition, and their use in generating insertion mutants via secondary transposition events The efficiency with which Tn5 and its derivatives transpose is influenced by the amount of transcription running into the ends of the Tn from flanking chromosomal promoters (Sasakawa et .0.1.,1982), and hence the frequency of TnblaM transposition will depend on its precise location within a chromosome. Amongst the JM101F- derivatives that contained TnblaM in the chromosome but did not encode BIaM fusion proteins (section b), some consistently yielded a few colonies when inoculated at high cell density (by patching) onto agar containing Ap (at concentrations exceeding its single cell MIC). Such Ap R colonies arise
when secondary transposition events occur that fuse the modified BIaM coding region in-frame to portions of genes encoding extracytoplasmic domains of proteins. Hence, the number of colonies obtained by patching different isolates onto agar containing Ap provides a guide to the relative frequencies of TnblaM transposition in each isolate. To identify the most effective TnblaM donors, aliquots of liquid cultures of different isolates were plated onto L agar containing 25/~g Ap/ml. The numbers of colonies obtained varied over a 100-fold range for different isolates, with the strain JBSI70 yielding the most (approx. 5000 Ap R colonies/ml). The ability to select large number of Ap l~ derivatives of JBS 170 suggests that the strategy of using an efficient TnblaM donor strain, coupled with an appropriate selection, should enable the recovery of even very rare types of TnblaM insertion mutants.
(d) The direct selection of isolates in which BlaM is fused to extracytoplasmic portions of highly abundant Escherickia coli gene products For any fusion in which the BlaM portion is translocared, the level of Ap R it confers on a cell is proportional to the amount of BIaM reaching the periplasm (BroomeSmith et al., 1989). Therefore, by selecting for derivatives of JBS 170 on agar containing increasing concentrations of Ap, it should, in theory, be possible to obtain the products of secondary transposition events in which BlaM is fused to extracytoplasmic portions of increasingly abundant proteins. To demonstrate the ease with which the products of rare TnblaM transposition events could be selected, and the feasibility of selectively isolating blaM fusions to poorly TABLE I Direct selection of Ap R derivatives of JBSI70 encoding BIaM fusion proteins Concentration of Ap (ttg/ml) used to select Ap R derivatives of JBS 170
Number of Ap R colonies obtained per ml JBS170 ~
Frequency of Ap R JB S 170 derivatives b
10 25 50 100 250 500 I000
8.4 x 4.8 x 1.9 x 1.2 x 2.1 x 39 6
1.1 × 6.0 × 2.4 x 1.5 × 2.6x 4.9 x 7.5 x
103 103 103 103 102
10- s 10- 6 10 - 6 10 - 6 10 - 7 10- s 10- 9
a ApR derivatives of JBS170 were selected by plating aliquots of a culture of JBS170, that had been grown overnight at 37°C in L broth, onto L agar containing Ap. Prior to plating the culture was sedimented and resuspended in fresh L broth, to remove Bla from the medium. The growth of bacteria was examined after overnight incubation at 37°C. b Ratio of numbers of colonies on plates in the presence vs. absence of Ap.
25 and highly expressed genes, aliquots of JBS 170 were plated onto agar containing increasing concentrations of Ap. As shown in Table I, derivatives encoding fusion proteins that conferred resistance to increasingly high concentrations of Ap occurred with decreasing frequencies, and although relatively rare, derivatives that were resistant to as much as 1 mg Ap/ml could be readily obtained. Five independent derivatives of JBS170 that had been selected on the basis of their resistance to lysis by 1 mg Ap/ml were characterised further. The MICs of Ap for single cells were determined by spotting 4pl of a 1:105 dilution of an overnight culture (about 40 bacteria) onto agar containing doubling concentrations of Ap. The values obtained were 2mg Ap/ml for JBSI54, JBS162 and JBS 169, and 4 mg Ap/ml for JBSI58 and JBS164, whilst that for the isogenic strain JM101F- [pBR322] was 8 mg Ap/ml. Western blotting revealed that, as expected from their very high single cell MICs for Ap, each of these isolates produces highly abundant BlaM fusion proteins (Fig. 3). It should be noted that, although the strategy of identifying an efficient TnblaM donor strain and using this to generate BIaM fusions via secondary transposition events constitutes a technically facile method for generating fusions and selecting even very rare insertion mutants, the chromosomes of isolates in which secondary transposition
kDa
a
b
c
d
e
f
Fig. 3. Detection of BIaM fusion proteins in highly Ap R J M I 0 1 F - TnblaM derivatives. Strains were grown at 37°C and cell lysates were fractionated by 0.1% SDS- 10 % PAGE (Spratt, 1977); proteins were blotted onto nitrocellulose. Blots were probed with rabbit antiserum to TEM Bla (used at a dilution of 1/1000). Horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) was used as second antibody at a dilution of 1/1000 and the blots were developed for 2 rain with 0.4 mg/ml 3,3'diaminobenzidine tetrahydrochloride and 0.012 % hydrogen peroxide. The strains were (a)JM101F-[pBR322] (encoding wild-type Bla); (b) IBS 158; (e) JBS 162; (d) JB S 169; (e) JB S 154; (f) JB S 164. The sizes of marker proteins (in kDa) are indicated by arrows.
events have occurred may contain a copy of TnbloM at the original, non-fusion, site as well as at the new site. Hence, transduction of the desired fusion gene into a clean genetic background is advisable before further genetic analysis of such insertion mutants.
(e) Delivery of TnblaM into the chromosomes of other Gram- bacteria. A variety of vectors have been developed for the delivery of Tn5 and its derivatives into the chromosomes of different Gram- bacteria. Usually these comprise plasmids or bacteriophage carrying the Tn that can be efficiently introduced into the target bacterial species but cannot replicate therein. Bacteriophage ;t derivatives carrying the transposon but deleted for the art site and with amber mutations in the O and P genes (rendering them incapable of integration or replication in suppressor-free strains) have proved highly effective for Tn mutagenesis orE. coli strains that are sensitive to phage A infection (Berg et al., 1975; Manoil and Beckwith, 1985). Although phage ). normally has a very restricted host range, this has been expanded to include a variety of Gram- bacteria by expressing the E. cog lamB gene (encoding the A receptor) in other bacterial species (Ludwig, 1987). Expression of lamb allows the ). phage to adsorb and inject its DNA, but not to replicate, in the new bacterial host. A phage A derivative carrying TnblaM was made by subcloning TnblaM from pJBS650 into INM627 (Fig. 2b). The efficacy of iTnblaM for mutagenesis of Erwinia carotovora strains expressing lamB (Salmond et al., 1986; Ellard et al., 1989) has recently been determined. Infection of E. carotovora with ATnblaM yielded Tn insertion mutants at similar frequencies to those obtained using ATn5. Moreover, following infection with ATnblaM, high efficiency Tn donor strains could be readily isolated, Ap R insertion mutants could be directly selected, and the insertion mutations could be transduced into new strains using Sp or Ap selection (G.P.C. Salmond, personal communication). (f) Conclusions (1) The transposon TnblaM, a Sp R derivative of TnS carrying blaM at its left end, was constructed. Following the delivery of TnblaM into the chromosome, or its transposition to new sites in the chromosome, bacterial mutants with TnblaM inserted into genes encoding cell envelope and secreted proteins can be directly selected as Ap R colonies. (2) TnblaM, like TnphoA, can be used to tag genes encoding cell envelope and secreted proteins. However, TnblaM has the advantage over TnphoA that mutants with insertions in genes encoding extracytoplasmic products can be obtained directly by Ap selection rather than indirectly
26
via phenotypic screening. Moreover, the Ap selection used with TnblaM is technically facile to apply and can be finetuned to obtain blaM fusions to poorly or well-expressed genes. (3) Bacterial isolates in which TnblaM is inserted into the chromosome at sites from which it transposes at high frequency can be readily identified, and constitute useful donors for the generation of rare insertion mutants via secondary transposition events. (4) TnblaM insertion t~:,tations can be transduced into new genetic backgrounds, or s~-~clonedinto plasmids, using Sp and/or Ap selection. (5) Plasmid and bacteriophage Avectors for the delivery of TnblaM into the E. coil chromosome and into the chromosomes of Gram- bacteria expressing lamb were constructed. For TnblaM mutagenesis of other bacteria, these vectors provide a convenient source of TnblaM for subcloning into the appropriate Tn delivery vectors. ACKNOWLEDGEMENTS
J.K.B.-S. and M.T. were funded by an MRC Senior Fellowship and MRC project grant, respectively. We thank G.P.C. Salmond for communicating results prior to publication, and Drs. P. Meacock, D.E. Berg, and N.E. Murray for providing plasmids and bacteriophage. REFERENCES Berg, D.E. and Berg, C.M.: The prokaryotic transposable element Tn5. Biotechnology 1 (1983) 417-435, Berg, D.E., Davies, J,, Allet,B, and Rochaix, J,D.: Transposition of R factorgenes to bacteriophage lambda. Proc, Natl.Acad. Sei.USA 72 (1975) 3628-3632. Broome-Smith, J.K. and Spratt, B.G.: A vector for the construction of translational fusions to TEM/~-Iaetamase and the analysis of protein export signals and membrane protein topology. Gene 49 (1986) 341349, Broome-Smith, J.K., Bowler, L.D, and Spratt, B.G.: A simple method for maximizing yields of membrane and exported proteins expressed in Escherichia coll. Mol. Mierobiol. 3 (1989) 1813-1817, Broome-Smith, J.K., Tadayyon, M, and Zhang, Y.: ~-lactamase as a probe of membrane protein assembly and protein export. Mol. Microbiol. 4 (1990) 1637-1644. De Bruijn, F.J, and Lupski, J.R,: The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids. Gene 27 (1984) 131149.
Ellard, F.M., Cabello, A. and Salmcnd, G.P.C.: Bacteriophage ~.mediated transposon mutagenesis of phytopathogenie and epiphytic Erwinia species is strain dependent. Mol. Gen. Genet. 21 (1989) 491498. Hoffman, C. and Wright, A.: Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Prec. Natl. Aead. Sci. USA 82 (1985) 5107-5111. Holmes, D.S. and Quigley, M.: A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114 (1981) 193-197. Johnson, R.C. and Reznikoff, W.S.: DNA sequences at the ends of Tn5 required for transposition. Nature 304 (1983) 280-282. Lathe, R., Kieny, M.P., Skory, S. and Lecocq, J.-P.: Linker-tailing: unphosphorylated linker oligonucleotidesfor joining DNA termini. DNA 3 (1984) 173-182. Ludwig, R.A.: Gene tandem-mediated selection ofcoliphage ,t-receptive Agrobacterium, Pseudomonas and Rhizobium strains. Prec. Natl. Acad. Sei. USA 84 (1987) 3334-3338. Manoil, C. and Beekwith, J.R.: TnphoA: a transposon probe for protein export signals. Prec. Natl. Acad. Sci. USA 82 (1985) 8129-8133. Manoil, C. and Beckwith, J.R.: A genetic approach to analyzing membrane protein topology. Science 233 (1986) 1403-1408. Manoil, C., Mekalanos, J.J. and Beckwith, J.: Alkaline phosphatase fusions: sensors of subeeilular location. J. Bacterioi. 172 (1990) 515518. Meacock, P.A. and Cohen, S.N.: Genetic analysis ofthe inter-relationship between plasmid replication and incompatibility, Mol. Gen. Genet. 174 (1979) I35-147. Miller, J.H.: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY, 1972. Prentki, P. and Krisch, H.M.: In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29 0984) 303-313. Rothstein, S.J., Jorgensen, R.A., Yin, J.C.-P., Yong-Di, Z., Johnson, R.C. and Reznikoff, W.S.: Genetic organization of Tn5. Cold Spring Harbor Symp. Quant. Biol. 45 (1980) 99-105. Salmond, G.P.C., Hinton, J.C.D., Gill, D.R. and Perombelon, M.C.M.: Transposon mutagenesis of Erwinia using phage ~.vectors. Mok Gen. Genet. 203 (1986) 524-528. Sasakawa, C., Lowe, J.B., McDivitt, L. and Berg, D.E.: Control of transposen Tn5 transposition in Escherichia coll. Prec. Natl. Acad. Sci. USA 79 (1982) 7450-7454. Sasakawa, C., Carle, G.F. and Berg, D.E.: Sequences essential for transposition at the termini of ISS0. Prec. Natl. Acad. Sci. USA 80 (1983) 7293-7297. Sprau, B.G.: Properties of the penicillin-binding proteins of Escherichia coil K-12. Eur. J. Biochem. 72 (1977) 341-352. Spratt, B.G,. Hedge, P.J., te Heesen, S., Edelman, A. and Broome-Smith, J.K.: Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. Gene 41 (1986) 337-342. Zhang, Y. and Broome-Smith, J.K.: Identification of amino acid sequences that can function as translocators of fl-laetamase in Escherichia coil. Mol. Microbiol. 3 (1989) 1361-1369. Zhang, Y. and Broome-Smith, J.K.: Correct insertion era simple eukaryotie plasma membrane protein into the cytoplasmic membrane of Escherichia coll. Gene 96 (1990) 51-57.
21
GENE 06299
TnblaM: a transposon for directly tagging bacterial genes encoding cell envelope and secreted proteins (Ampicillin; spectinomycin; protein fusions;//-lactamase; mutagenesis; TnphoA; membrane proteins; exported proteins; recombinant DNA)
M. Tadayyonand JennyK. Broome-Smith Microbial Genetics Group, School of Biological Sciences, University of Sussex, Falmer, Brighton BNI 9Q,G (U.K.) Received by P.A. Manning: 19 August 1991 Revised/Accepted: 11 November/13 November 1991 Received at publishers: 5 December 1991
SUMMARY
A transposon, TnblaM, designed for the direct selection of bacterial mutants with insertions in genes encoding cell envelope and secreted proteins, was constructed and subcloned into plasmid and bacteriophage ;. delivery vectors. TnblaM is a spectinomycin-resistantderivative of Tn5 with an unexpressed open reading frame encoding mature//-lactamase (BIaM) at its left end. Therefore, when it inserts into genes in the correct orientation and reading frame, gene fusions encoding hybrid proteins are generated. By introducing TnblaM into bacterial cells and selecting ampicillin-resistant (Ap R) colonies, the subset of isolates producing extracytoplasmic BIaM, and hence containing TnblaM inserted in genes encoding secreted proteins and cell envelope proteins, can be directly selected. TnblaM, fike TnphoA, can therefore be used to preferentially mutagenise genes encoding extracytoplasmic proteins, but is has the advantage over TnphoA that the desired mutants can be isolated by direct selection (as Ap R colonies) rather than by phenotypie screening. Isolates in which TnblaM occupies sites in the chromosome from which it can transpose at high frequency are readily identifiable, and constitute TnblaM donors, with which to simply and efficiently generate rare types of insertion mutants. Moreover, the Ap R selection that is used with TnblaM can be fine-tuned to obtain blaM fusions to poorly or well-expressed genes.
INTRODUCTION
Gene fusion studies have proved immensely valuable in the analysis of gene expression and protein structure and Correspondence to: Dr. J.K. Broome-Smith, Microbial Genetics Group, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG (U.K.) Tel. (44-0273)606755; Fax (44-0273)678433. Abbreviations: Ap, ampicillin;Bla,//-lactamase; BlaM, mature portion of Bla; blaM, 5'-truncated bin gene coding for BIaM; bp, base pair(s); IS, insertion sequence; kb, kilobase(s) or 1000bp; Km, kanamycin; MIC, minimum inhibitory concentration; nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide-gelelectrophoresis; R, resistance/resistant; s, sensitivity/sensitive;Sp, spectinomycin; Tc, tetracycline; TEM, specifies the source of Bla; Tn, transposon; XP, 5-bromo-4-chloro-3-indolyl phosphate; [ ], denotes plasmid-carrier state.
function. Two translational fusion systems have been developed specifically for analysing the organization of proteins within the bacterial cytoplasmic membrane and identifying bacterial export signals. In both systems hybrid proteins are generated by fusing the coding regions for reporters that display location-specific phenotypes to progressively truncated forms of the target genes. In the first system the mature form of alkaline phosphatase is used as the reporter. Only extracytoplasmic forms of alkaline phosphatase are enzymatically active. Therefore, by plating cells onto agar containing XP, a chromogenic substrate for alkaline phosphatase, all isolates in which alkaline phosphatase has been fused to translocated portions of proteins can be identified as blue-coloured colonies. Hence this reporter provides a way of screening for cytoplasmic memhrane export signals (Manoil and Beckwith, 1985). In the
22
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d ~
Tnbla/'f E clB57
Fig. 2
Bc/B
Tnbla/'/
Fig. 1
o~-
,t s~
Fig. 1. Construction and structure of TnblaM. Only the relevant restriction sites are shown. (a) Construction of TnblaM. Unless otherwise stated, the suppressor=free strain E. coil HB2151 (Alac-pro, thi, [F', proA*B ÷, lacI°ZAMIS, traD36]) was used for transformations and was grown in L broth or on L agar (Miller, 1972) supplemented with 50/Jg Km/ml, 20 #g Sp/ml, 20 pg Tc/ml or Ap (variable concentrations) as required. A derivative of pBR322 with Tn5 inserted at nt 31-39 was provided by Dr. D.E. Berg, Washington University, St. Louis, MO. This plasmid was digested with BamHI and the fragment carrying ISSOR was inserted into the BamHI site of pBS~- (Spratt et al., 1956) to produce pJBS647. Plasmid pJBS647 DNA prepared from a dam- strain was linearised with Bcll, and ligated to the 2-kb BamHl fragment encoding Sp R (the ~ cassette) obtained from pHP45f~ (Prentki and Krisch, 1984) to produce pJBS648. Plasmid pJBS646 contains blaM inserted as a PruII-AhaIII fragment into the HincII site of pteHl9 (Spratt et al., 1986) and encodes a cytoplasmic form of BlaM (Broome~Smith and Spratt, 1986). This plasmid was linearised by partial AhaIII digestion and ligated to the end-filled BamHl fragment from pJBS648 containing the Sp R gene, to yield pJBS649 in which blaM precedes the Sp R gene and all of the ISSOR sequences that are essential for Tr6 transposition. Finally, pBRG700, provided by Dr. D.E. Berg, was used as a source of an Alul-BamHi fragment
23 second system the mature form of BIaM is used as the reporter. In this case translational fusions to both cytoplasmic and extracytoplasmie portions of target proteins can be readily identified since isolates producing BIaM fusion proteins can grow when plated at high inocula onto agar containing Ap. However, only translocated forms of BIaM are able to protect individual cells against lysis by Ap. Therefore, isolates in which the BIaM moiety is extracytoplasmic can be distinguished from those in which it is cytoplasmic by their ability to survive and form colonies when plated at high dilution onto agar containing Ap (Broome-Smith and Spratt, 1986). Moreover, by selecting Ap R colonies the subset of isolates in which BlaM is fused to extracytoplasmic domains of proteins can be directly isolated. Hence when BlaM is used as the reporter, cytoplasmic membrane export signals can be directly selected (e.g., see Zhang and Broome-Smith, 1989). Plasmid vectors for the construction of alkaline phosphatase fusions to cloned genes in vitro, and a transposon, TnphoA, for the insertion of the mature alkaline phosphatase coding region into plasmid and bacterial chromosomes in vivo, have been constructed and widely used to screen for genes containing export signals (Hoffman and Wright, 1985; Manoil and Beckwith, 1985; 1986; Manoil et al., 1989). Plasmid vectors for the generation of blaM fusions in vitro to cloned bacterial and eukaryotic genes expressed in E. colt have also been described and used to analyse membrane protein topology (Broome-Smith and Spratt, 1986; Zhang and Broome-Smith, 1990; reviewed in Broome-Smith et al., 1990). We report here the construction of the Tn5 derivative TnblaM and show how it can be introduced into Gram- bacteria and used to directly select mutants with TnblaM insertions in genes encoding cell envelope and secreted proteins.
RESULTS AND DISCUSSION
(a) Construction of TnblaM Tn5 is a composite Tn in which inverted repeats of the smaller transposable elements IS.S0L and ISSOR bracket a central region that encodes Km R (Berg and Berg, 1983). Genetic analysis has revealed that only the transposase gene of ISSOR and the outermost 19 bp of each IS are required for the efficient transposition of any intervening sequences (Rothstein et al., 1980; Sasakawa et al., 1983; Johnson and Reznikoff, 1983). Moreover, Tn5 and its derivatives are able to transpose in a broad range of Grambacteria and display a relatively low level of insertional specificity, making them ideal elements with which to mutagenise the genomes of many different bacteria (reviewed in De Bruijn and Lupski, 1984). The strategy used to make TnblaM was to fuse the outer terminus of ISSOL to the start of blaM, such that the ORF encoding BIaM was extended back to the outer end of ISSOL, and to insert a S p R determinant between the modified BlaM-coding region, and the transposase gene and outer terminus oflSSOR. The construction ofpJBS650 that carries TnblaM, and the relationship of TnblaM to TnS, are outlined in Fig. 1. (b) Delivery of TnblaM into the Escherichia coli chromosome A plasmid vector for delivering TnblaM into the E. colt chromosome was made by subcloning the transposon from pJBS650 into a pSC101 derivative that is temperaturesensitive for replication in E. colt (Fig. 2a). DNA of the resultant plasmid, pJBS651, was used to transform the strain JMI01F- (Aiac-pro, tht, supE) to Tc R at 30°C, and a transformant was purified at 30°C on L agar containing
containing the outer 19 bp of ISSOL (Sasakawa et al., 1983) and this fragment was inserted between the Smal and BamHI sites of pJBS649, yielding pJBS650 in which the blaM ORF is N-terminally extended, back through the outer terminus of IS50L. Therefore, pJBS650 contains a novel Tn, TnblaM, that possesses all of the sequences necessary for the efficient transposition of the Spa marker and the modified BlaM-coding region. IS sequences are boxed. (b) The relationship of TnblaM to TnS. The stippled and wavy shading denote the BlaM-coding region and fl cassette, respectively. The sequence from the left end of TnblaM to the third codon of the BlaM coding region is: 5'-CTG ACT CTT ATA CAC AAG TCG GGA TCC GTC CTG CGT CAC CCA GAA... Leu Thr Leu Ile His Lys Ser Gly Ser Val Leu Arg His Pro Glu... +1 +2 +3 Abbreviations: A, Ahalll; B, BamHl; Be, BclI; E, EcoRI; EF, end-filled; H, Hindlll; lacpo, lac promoter and operator; P, Pstl; RV, EcoRV; S, Sail; Sin, Sinai; Sph, Sphl; StrR, streptomycin-resistance marker. Symbols × indicate cleavage by restriction enzymes. Fig~ 2. Plasmid and bacteriophage vectors carrying TnblaM. Only the relevant restriction sites are shown. (a) Construction ofpJBS651. Plasmid pPM 121, a pSCI01 derivative that is temperature-sensitive for replication in E. colt (Meacock and Cohen, 1979)was obtained from Dr. P.A. Meacock, University of Leicester, U.K. TnblaM was cloned into pPMl21 by digesting pJBS650 with EcoRI + EcoRV, and ligating the digested DNA to pPM 121 DNA that had been cut with EcoRI + Smal, yielding pJBS651. (b) Construction of ,tTnblaM. Plasmid pJBS650 DNA was digested with EcoRV and an EcoRI linker was added by linker tailing (Lathe et al., 1984) to produce pJBS652. Phage ANM627 has the genotype A(srlA 1.2) c1857 srl,[4 ° nin5 srl,~5 ° Sam7 and was obtained from Dr. N.E. Murray, Department of Molecular Biology, University of Edinburgh, U.K. DNA of ANM627 was digested with EcoRl, and the left and right arms of the bacteriophage were purified and ligated to pJBS652 DNA that had been digested with EcoRl, to produce ,[TnblaM. Abbreviations as in Fig. 1 legend.
24 20 pg Tc/ml and 20 #g Sp/ml. To obtain E. coil derivatives carrying TnblaM in the chromosome, JMI01F- [pJBS651 ] was then grown for approx. 20 generations at 42°C in the absence of any antibiotics (to dilute out the plasmid), and Sp R isolates were selected. Less than 1% of these were Tc R, and examination of the DNA content of cell lysates (Holmes and Quigley, 1981) revealed that they did not contain any pJBS651 plasmid DNA (data not shown). Most (approx. 70%) of the above isolates failed to form an area of complete growth when inoculated at high cell density (by toothpicking colonies) onto agar containing 10 #g Ap/ml, suggesting that TnblaM had inserted into the chromosome such that its BIaM coding region was unexpressed. However, the remaining isolates formed an area of complete growth in this 'patch test', indicating that blaM was either fused in-frame to an N-terminal portion of a chromosomal gene, or that it was fused out-of-frame to aa N-terminal portion of a relatively well-expressed gene (diEcussed in Broome-Smith and Spratt, 1986). Isolates in which transposition of TnblaM into the chromosome had resulted in the fusion of BIaM to extracytoplasmic portions of E. coil gene products were directly selected by plating aliquots of the culture of JM101F-[pJBS651] that had been grown in antibioticfree medium at 42°C (see above) onto L agar containing Ap. When Ap was used at a concentration of 10 #g/ml (i.e., at twice the concentration that lyses non-fl-lactamase producing cells of E. coil or cells producing cytoplasmic forms of//-lactamase), 1 in 4 × 105 cells grew and formed colonies. Several of these isolates were purified and then plated at high dilution onto agar containing 5 ~tg Ap/ml. Individual cells of each isolate were able to grow and form colonies under these conditions, confirming that they produced extracytoplasmic forms of Bin. Hence, by combining a method for delivering TnblaM into the E. coil chromosome with selection for Ap R colonies, mutants of E. coli with TnblaM inserted in genes encoding extracytoplasmic proteins were directly selected.
(e) Identification of strains that display efficient TnblaM transposition, and their use in generating insertion mutants via secondary transposition events The efficiency with which Tn5 and its derivatives transpose is influenced by the amount of transcription running into the ends of the Tn from flanking chromosomal promoters (Sasakawa et .0.1.,1982), and hence the frequency of TnblaM transposition will depend on its precise location within a chromosome. Amongst the JM101F- derivatives that contained TnblaM in the chromosome but did not encode BIaM fusion proteins (section b), some consistently yielded a few colonies when inoculated at high cell density (by patching) onto agar containing Ap (at concentrations exceeding its single cell MIC). Such Ap R colonies arise
when secondary transposition events occur that fuse the modified BIaM coding region in-frame to portions of genes encoding extracytoplasmic domains of proteins. Hence, the number of colonies obtained by patching different isolates onto agar containing Ap provides a guide to the relative frequencies of TnblaM transposition in each isolate. To identify the most effective TnblaM donors, aliquots of liquid cultures of different isolates were plated onto L agar containing 25/~g Ap/ml. The numbers of colonies obtained varied over a 100-fold range for different isolates, with the strain JBSI70 yielding the most (approx. 5000 Ap R colonies/ml). The ability to select large number of Ap l~ derivatives of JBS 170 suggests that the strategy of using an efficient TnblaM donor strain, coupled with an appropriate selection, should enable the recovery of even very rare types of TnblaM insertion mutants.
(d) The direct selection of isolates in which BlaM is fused to extracytoplasmic portions of highly abundant Escherickia coli gene products For any fusion in which the BlaM portion is translocared, the level of Ap R it confers on a cell is proportional to the amount of BIaM reaching the periplasm (BroomeSmith et al., 1989). Therefore, by selecting for derivatives of JBS 170 on agar containing increasing concentrations of Ap, it should, in theory, be possible to obtain the products of secondary transposition events in which BlaM is fused to extracytoplasmic portions of increasingly abundant proteins. To demonstrate the ease with which the products of rare TnblaM transposition events could be selected, and the feasibility of selectively isolating blaM fusions to poorly TABLE I Direct selection of Ap R derivatives of JBSI70 encoding BIaM fusion proteins Concentration of Ap (ttg/ml) used to select Ap R derivatives of JBS 170
Number of Ap R colonies obtained per ml JBS170 ~
Frequency of Ap R JB S 170 derivatives b
10 25 50 100 250 500 I000
8.4 x 4.8 x 1.9 x 1.2 x 2.1 x 39 6
1.1 × 6.0 × 2.4 x 1.5 × 2.6x 4.9 x 7.5 x
103 103 103 103 102
10- s 10- 6 10 - 6 10 - 6 10 - 7 10- s 10- 9
a ApR derivatives of JBS170 were selected by plating aliquots of a culture of JBS170, that had been grown overnight at 37°C in L broth, onto L agar containing Ap. Prior to plating the culture was sedimented and resuspended in fresh L broth, to remove Bla from the medium. The growth of bacteria was examined after overnight incubation at 37°C. b Ratio of numbers of colonies on plates in the presence vs. absence of Ap.
25 and highly expressed genes, aliquots of JBS 170 were plated onto agar containing increasing concentrations of Ap. As shown in Table I, derivatives encoding fusion proteins that conferred resistance to increasingly high concentrations of Ap occurred with decreasing frequencies, and although relatively rare, derivatives that were resistant to as much as 1 mg Ap/ml could be readily obtained. Five independent derivatives of JBS170 that had been selected on the basis of their resistance to lysis by 1 mg Ap/ml were characterised further. The MICs of Ap for single cells were determined by spotting 4pl of a 1:105 dilution of an overnight culture (about 40 bacteria) onto agar containing doubling concentrations of Ap. The values obtained were 2mg Ap/ml for JBSI54, JBS162 and JBS 169, and 4 mg Ap/ml for JBSI58 and JBS164, whilst that for the isogenic strain JM101F- [pBR322] was 8 mg Ap/ml. Western blotting revealed that, as expected from their very high single cell MICs for Ap, each of these isolates produces highly abundant BlaM fusion proteins (Fig. 3). It should be noted that, although the strategy of identifying an efficient TnblaM donor strain and using this to generate BIaM fusions via secondary transposition events constitutes a technically facile method for generating fusions and selecting even very rare insertion mutants, the chromosomes of isolates in which secondary transposition
kDa
a
b
c
d
e
f
Fig. 3. Detection of BIaM fusion proteins in highly Ap R J M I 0 1 F - TnblaM derivatives. Strains were grown at 37°C and cell lysates were fractionated by 0.1% SDS- 10 % PAGE (Spratt, 1977); proteins were blotted onto nitrocellulose. Blots were probed with rabbit antiserum to TEM Bla (used at a dilution of 1/1000). Horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) was used as second antibody at a dilution of 1/1000 and the blots were developed for 2 rain with 0.4 mg/ml 3,3'diaminobenzidine tetrahydrochloride and 0.012 % hydrogen peroxide. The strains were (a)JM101F-[pBR322] (encoding wild-type Bla); (b) IBS 158; (e) JBS 162; (d) JB S 169; (e) JB S 154; (f) JB S 164. The sizes of marker proteins (in kDa) are indicated by arrows.
events have occurred may contain a copy of TnbloM at the original, non-fusion, site as well as at the new site. Hence, transduction of the desired fusion gene into a clean genetic background is advisable before further genetic analysis of such insertion mutants.
(e) Delivery of TnblaM into the chromosomes of other Gram- bacteria. A variety of vectors have been developed for the delivery of Tn5 and its derivatives into the chromosomes of different Gram- bacteria. Usually these comprise plasmids or bacteriophage carrying the Tn that can be efficiently introduced into the target bacterial species but cannot replicate therein. Bacteriophage ;t derivatives carrying the transposon but deleted for the art site and with amber mutations in the O and P genes (rendering them incapable of integration or replication in suppressor-free strains) have proved highly effective for Tn mutagenesis orE. coli strains that are sensitive to phage A infection (Berg et al., 1975; Manoil and Beckwith, 1985). Although phage ). normally has a very restricted host range, this has been expanded to include a variety of Gram- bacteria by expressing the E. cog lamB gene (encoding the A receptor) in other bacterial species (Ludwig, 1987). Expression of lamb allows the ). phage to adsorb and inject its DNA, but not to replicate, in the new bacterial host. A phage A derivative carrying TnblaM was made by subcloning TnblaM from pJBS650 into INM627 (Fig. 2b). The efficacy of iTnblaM for mutagenesis of Erwinia carotovora strains expressing lamB (Salmond et al., 1986; Ellard et al., 1989) has recently been determined. Infection of E. carotovora with ATnblaM yielded Tn insertion mutants at similar frequencies to those obtained using ATn5. Moreover, following infection with ATnblaM, high efficiency Tn donor strains could be readily isolated, Ap R insertion mutants could be directly selected, and the insertion mutations could be transduced into new strains using Sp or Ap selection (G.P.C. Salmond, personal communication). (f) Conclusions (1) The transposon TnblaM, a Sp R derivative of TnS carrying blaM at its left end, was constructed. Following the delivery of TnblaM into the chromosome, or its transposition to new sites in the chromosome, bacterial mutants with TnblaM inserted into genes encoding cell envelope and secreted proteins can be directly selected as Ap R colonies. (2) TnblaM, like TnphoA, can be used to tag genes encoding cell envelope and secreted proteins. However, TnblaM has the advantage over TnphoA that mutants with insertions in genes encoding extracytoplasmic products can be obtained directly by Ap selection rather than indirectly
26
via phenotypic screening. Moreover, the Ap selection used with TnblaM is technically facile to apply and can be finetuned to obtain blaM fusions to poorly or well-expressed genes. (3) Bacterial isolates in which TnblaM is inserted into the chromosome at sites from which it transposes at high frequency can be readily identified, and constitute useful donors for the generation of rare insertion mutants via secondary transposition events. (4) TnblaM insertion t~:,tations can be transduced into new genetic backgrounds, or s~-~clonedinto plasmids, using Sp and/or Ap selection. (5) Plasmid and bacteriophage Avectors for the delivery of TnblaM into the E. coil chromosome and into the chromosomes of Gram- bacteria expressing lamb were constructed. For TnblaM mutagenesis of other bacteria, these vectors provide a convenient source of TnblaM for subcloning into the appropriate Tn delivery vectors. ACKNOWLEDGEMENTS
J.K.B.-S. and M.T. were funded by an MRC Senior Fellowship and MRC project grant, respectively. We thank G.P.C. Salmond for communicating results prior to publication, and Drs. P. Meacock, D.E. Berg, and N.E. Murray for providing plasmids and bacteriophage. REFERENCES Berg, D.E. and Berg, C.M.: The prokaryotic transposable element Tn5. Biotechnology 1 (1983) 417-435, Berg, D.E., Davies, J,, Allet,B, and Rochaix, J,D.: Transposition of R factorgenes to bacteriophage lambda. Proc, Natl.Acad. Sei.USA 72 (1975) 3628-3632. Broome-Smith, J.K. and Spratt, B.G.: A vector for the construction of translational fusions to TEM/~-Iaetamase and the analysis of protein export signals and membrane protein topology. Gene 49 (1986) 341349, Broome-Smith, J.K., Bowler, L.D, and Spratt, B.G.: A simple method for maximizing yields of membrane and exported proteins expressed in Escherichia coll. Mol. Mierobiol. 3 (1989) 1813-1817, Broome-Smith, J.K., Tadayyon, M, and Zhang, Y.: ~-lactamase as a probe of membrane protein assembly and protein export. Mol. Microbiol. 4 (1990) 1637-1644. De Bruijn, F.J, and Lupski, J.R,: The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids. Gene 27 (1984) 131149.
Ellard, F.M., Cabello, A. and Salmcnd, G.P.C.: Bacteriophage ~.mediated transposon mutagenesis of phytopathogenie and epiphytic Erwinia species is strain dependent. Mol. Gen. Genet. 21 (1989) 491498. Hoffman, C. and Wright, A.: Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Prec. Natl. Aead. Sci. USA 82 (1985) 5107-5111. Holmes, D.S. and Quigley, M.: A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114 (1981) 193-197. Johnson, R.C. and Reznikoff, W.S.: DNA sequences at the ends of Tn5 required for transposition. Nature 304 (1983) 280-282. Lathe, R., Kieny, M.P., Skory, S. and Lecocq, J.-P.: Linker-tailing: unphosphorylated linker oligonucleotidesfor joining DNA termini. DNA 3 (1984) 173-182. Ludwig, R.A.: Gene tandem-mediated selection ofcoliphage ,t-receptive Agrobacterium, Pseudomonas and Rhizobium strains. Prec. Natl. Acad. Sei. USA 84 (1987) 3334-3338. Manoil, C. and Beekwith, J.R.: TnphoA: a transposon probe for protein export signals. Prec. Natl. Acad. Sci. USA 82 (1985) 8129-8133. Manoil, C. and Beckwith, J.R.: A genetic approach to analyzing membrane protein topology. Science 233 (1986) 1403-1408. Manoil, C., Mekalanos, J.J. and Beckwith, J.: Alkaline phosphatase fusions: sensors of subeeilular location. J. Bacterioi. 172 (1990) 515518. Meacock, P.A. and Cohen, S.N.: Genetic analysis ofthe inter-relationship between plasmid replication and incompatibility, Mol. Gen. Genet. 174 (1979) I35-147. Miller, J.H.: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY, 1972. Prentki, P. and Krisch, H.M.: In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29 0984) 303-313. Rothstein, S.J., Jorgensen, R.A., Yin, J.C.-P., Yong-Di, Z., Johnson, R.C. and Reznikoff, W.S.: Genetic organization of Tn5. Cold Spring Harbor Symp. Quant. Biol. 45 (1980) 99-105. Salmond, G.P.C., Hinton, J.C.D., Gill, D.R. and Perombelon, M.C.M.: Transposon mutagenesis of Erwinia using phage ~.vectors. Mok Gen. Genet. 203 (1986) 524-528. Sasakawa, C., Lowe, J.B., McDivitt, L. and Berg, D.E.: Control of transposen Tn5 transposition in Escherichia coll. Prec. Natl. Acad. Sci. USA 79 (1982) 7450-7454. Sasakawa, C., Carle, G.F. and Berg, D.E.: Sequences essential for transposition at the termini of ISS0. Prec. Natl. Acad. Sci. USA 80 (1983) 7293-7297. Sprau, B.G.: Properties of the penicillin-binding proteins of Escherichia coil K-12. Eur. J. Biochem. 72 (1977) 341-352. Spratt, B.G,. Hedge, P.J., te Heesen, S., Edelman, A. and Broome-Smith, J.K.: Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. Gene 41 (1986) 337-342. Zhang, Y. and Broome-Smith, J.K.: Identification of amino acid sequences that can function as translocators of fl-laetamase in Escherichia coil. Mol. Microbiol. 3 (1989) 1361-1369. Zhang, Y. and Broome-Smith, J.K.: Correct insertion era simple eukaryotie plasma membrane protein into the cytoplasmic membrane of Escherichia coll. Gene 96 (1990) 51-57.
