A vector for the construction of translational fusions to TEM β-lactamase and the analysis of protein export signals and membrane protein topology

341

Gene, 49 (1986) 341-349 Elsevier GENE

01866

A vector for the construction of translational fusions to TEM /!I-lactamase and the analysis of protein export signals and membrane protein topology (Protein fusions; cellular location; fl phage origin; recombinant DNA; nucleotide sequencing; ampicillin resistance; protease digestion; transposon TnphoA; spheroplasts; immunoblotting)

Jenny K. Broome-Smith * and Brian G. Spratt Microbial Genetics Group, School of Biological Sciences, University of Sussex, Falmer, Brighton BNl Tel. (0273)678309 (Received

September

(Accepted

October

9QG (U.K.)

3rd, 1986) 8th, 1986)

SUMMARY

A plasmid vector, pJBS633, that facilitates the construction of translational fusions of genes of interest to the coding region of the mature form of TEM /I-lactamase has been developed. Transformants containing in-frame fusions can be identified by their ability to grow when plated at high inocula on agar containing ampicillin (Ap). The cellular location of the /?-lactamase moiety of the fusion proteins can then be determined since only those that direct the translocation of the /&lactamase across the cytoplasmic membrane to the periplasm result in the ability of individual cells of Escherichia coli to form isolated colonies in the presence of Ap. Conversely, those fusion proteins in which the /&lactamase moiety remains cytoplasmic do not protect individual cells against Ap. Transformants expressing the latter class of fusion proteins can, however, be identified when plated at high inocula since, as cells start to lyse, the cytoplasmic B-lactamase activity is released and provides Ap resistance to the surrounding cells. The vector contains the origin of replication of fl phage so that single-stranded plasmid DNA can be obtained in the appropriate orientation to allow sequencing across the fusion junction using a universal primer complementary to the start of the coding region of mature TEM B-lactamase. pJBS633 should be useful as a general vector for the construction of /I-lactamase fusions and, in particular, for the analysis of protein export signals and the determination of the organisation of proteins in the E. coli cytoplasmic membrane.

INTRODUCTION

In the Gram-negative bacterium E. coli all protein synthesis occurs in the cytoplasm, yet many proteins * To whom

correspondence

and

reprint

requests

should

be

addressed.

are ultimately localised to the non-cytoplasmic compartments - the cytoplasmic membrane, the periplasm, and the outer membrane. An understanding of the genetic information that enables proteins to be

1000 bp; Km, kanamycin; polyacrylamide tein; PMSF,

Abbreviations:

Ap,

coding for the mature pax(s);

blaM, 5’-truncated

ampicillin;

portion of the /?-lactamase

CAT, chloramphenicol

acetyltransferase:

bla gene

(ApR); bp, base

A, deletion; kb,

sensitivity;

carrier 0378-I 119/86/$03.50

0

1986 Elsewer

Saence

Publishers

B.V. (Biomedical

ss, single stranded;

Dwision)

fluoride;

SDS, sodium

et al., 1984);

pro-

R, resistance;

‘,

dodecylsulphate;

TEM, specifies the source of #?-lactamase

see Kadonaga state.

PAGE,

PBP, penicillin-binding

phenyl methyl sulphonyl

Tc, tetracycline; RTEM,

on’,origin of DNA replication;

gel electrophoresis;

[ 1, designates

(also

plasmid-

342

exported, and is responsible for their accurate targeting to non-c~oplasmi~ comp~ents, is of basic biological interest. Until recently much of our understanding of this process was derived from work on protein fusions in which N-terminal portions of an exported protein were fused to the enzyme ~-g~a~tosidase (Benson et al., 198.5; Silhavy and Beckwith, 1985; Silhavy et al., 1984). However, although /I-galactosidase fusion technology has been very successful for the genetic analysis of signal peptides (Silhavy et al., 1984) and the isolation of mutants that may define cellular components of the protein export machinery (Oliver, 1985), the recent realisation that /?-galactosidase cannot be translocated across the cytoplasmic membrane of E. cd imposes severe limitations on the use of B-galactosidase fusions for many studies of protein export (Tommassen et al., 1985). Hoffman and Wright (1985) have suggested using the mature form of the periplasmic enzyme alkaline phosphatase as an alternative to /I-galactosidase. They showed that mature alkaline phosphatase could be translocated across the cytoplasmic membrane provided it was fused to an N-terminal portion of a secreted protein that included an intact signal sequence. A transposon, TnphoA, was subsequently constructed that facilitates the fusion of the coding region of the mature form of alkaline phosphatase to other genes (Manoil and Beckwith, 1985). Since alkaline phosphatase only possesses enzymatic activity after translocation across the cytoplasmic membrane, the use of a chromogenic substrate for the enzyme allows the detection of only those colonies in which the tr~sposition of TnphuA has resulted in the expression of a fusion protein that can translocate the alkaline phosphatase moiety into the periplasm. TnphoA therefore provides a valuable tool for the study of the process of protein export and for the genetic analysis of the topology of cytoplasmic membrane proteins (Manoil and Be&with, 1985). We anticipated that the use of TEM &lactamase might be preferable to that of alkaline phosphatase since in the latter system only those fusions that tramlocate alkaline phosphatase across the cytoplasmic membrane are identified, whereas with /I-lactamase it should be possible to identify all in-frame fusions and subsequently to determine whether or not the /?-lactamase moiety of the fusion protein has been translocated. We describe here the

construction of a plasmid vector, pJBS633, that can be used to make fusions of any gene to the coding region of mature p-lactamase, and that facilitates the rapid determination of the nucleotide sequence of the fusion junctions. The vector appears to provide a simple and reliable tool for the analysis of protein export signals and the topology of E. coli c~oplasmic membrane proteins.

MATERIALS AND METHODS

(a) Bacterial strains and plasmids E. coZiHB2151 (&c-pro, tZzi,sup’ [F’,proA+ B+, ZacZQZldM15, traD36]) was used in all experiments and was obtained from Anglian Biotechnology Ltd, Colchester, Essex (U.K.). L broth or L agar (Miller, 1972) was used in most experiments and was supplemented with 50 pg Km/ml, 10~8 Tc/ml or Ap (variable concentrations; see Table I) as required. Growth of bacteriophage fl-infected cultures was in 2TY broth (Miller, 1972). Plasmid pTG2 is a derivative of pBR322 that has a BstEII site straddling the junction between the coding region of the signal peptide and mature part of TEM p-lactamase (Kadonaga et al., 1984). (b) Preparation of plasmid single-stranded DNA and nucleotide sequencing

Single-stranded (ss) DNA was obtained from pJBS633 derivatives by superinfection with phage fl, variant IRl, as described by Dente et al. (1983) and the nucleotide sequence across the /?-lactamase fusion junctions was determined by the dideoxy method (Biggin et al., 1983) using a primer (dCTCGTGCACCCAACTGA) that was complementary to codons 14-18 of the mature form of TEM /$lactamase and which primed sequencing across the PvuII site towards the TcR gene. (c) Identification frame fusions

of transformants

producing

in-

The preliminary screening for transformants that contained in-frame fusions of a gene of interest to the coding region of the mature form of TEM /?lactamase was carried out by toothpicking colonies

343

onto L agar containing 200 pg Ap/ml and incubating the plates overnight at 37°C. Using this ‘patch test’ those transformants expressing in-frame fusions gave strong growth across the patch whereas those with out-of-frame fusions failed to grow or grew weakly across only part of the patch. The patch test can be applied to large numbers of transformants and identifies all in-frame fusions but can result in the misclassification of some out-of-frame fusions (See RESULTS AND DISCUSSION, section e). A more reliable, but more time-consuming, ‘spotting test’ was applied to those transformants that appeared from the patch test to have in-frame fusions. Six-p1 amounts of overnight cultures of tr~sfo~~ts were spotted on L agar plates containing 200 pg Ap/ml and the plates were incubated overnight at 37°C. Transformants with in-frame fusions showed strong growth whereas those with out-of-frame fusions failed to grow. It should be noted that the levels of Ap used in both the patch test and the spotting test can probably be increased when studying fusions to strongly expressed genes and may need to be decreased when working with genes that are very poorly expressed.

Overnight cultures of strain HB215 1 carrying pJBS633 derivatives were diluted 1: lo5 in L broth and 4 ~1 amounts (containing about 40 bacteria) were spotted on L agar plates containing a range of concentrations of Ap (see Table I). The growth of the bacteria was examined after overnight incubation at 37°C.

catalyses the hydrolysis of @actam antibiotics such as Ap and in its periplasmic location can efficiently protect a cell against lysis by fl-lactams since the incoming antibiotic is degraded before it can inactivate the killing targets (essential enzymes of peptidoglyc~ synthesis) that are located in the cytoplasmic membrane (Spratt, 1983). Deletion of the region encoding the signal peptide of /3-lactamase results in the production of a cytoplasmic form ofthe mature enzyme (Kadonaga et al., 1984). The intemalised enzyme affords no protection to a single cell against lysis by fi-lactams since the ~tibiotic is not prevented from reaching its killing targets in the cytoplasmic membrane. However, the intemalised enzyme does provide protection against the antibiotic action of Ap when the cells are plated at a high inoculum (e.g., patching colonies with a toothpick) since, following exposure to Ap, some cells in the population start to lyse and release the intemalised @-lactamase into the medium where it hydrolyses the Ap and thereby protects nei~bou~ng cells in the population from being killed (Kadonaga and Knowles, 1985). Provided fi-lactamase fusion proteins consistently retain enzymatic activity it should be possible to use the above ‘patch test’ to identify all of those cells in which a target gene has been fused in-frame to the coding region of mature /&lactamase, irrespective of the cellular location of the resultant fusion protein. The ability, or inability, of the fusion protein to protect single cells against lysis by Ap should then indicate whether the /%lactamase moiety of the fusion protein has been translocated across the cytoplasmic membrane or whether it has remained in the cytoplasm.

RESULTS AND DISCUSSION

(b) Construction of a vector, pJRS633, for making translational fusions to mature TEM /Uactamase

(d) Measurement of the level of ampicillin resistance of single cells

(a) Rationale for the choice of TEM &lactamase as a probe for studying the process of protein export and membrane protein topology

/?-Lactamase, like alkaline phosphatase, is initially synthesised as a preprotein with an N-terminal signal peptide that is proteolytically removed during translocation of the protein across the cytoplasmic membrane to yield the mature periplasmic enzyme (Koshland and Botstein, 1982). fi-Lactamase

The construction of pJBS633 is outlined in Fig. 1. The BstEII site in pTG2, which is at the junction of the coding region for the signal peptide and mature part of TEM /.?-lactamase, was converted to a more convenient blunt-end cloning site by end-filling with T4 DNA polymerase and PvuII linker addition to produce pJBS63 1. This results in the introduction of an additional two codons between the PvuII site and the first codon of mature @-lactamase (Fig. 1). The PvtlII fragment of pJBS631 ~ont~ning the coding

344

A”ll (11

j

pJBSi3l 6

OKb

PVUll

x PVUII \

EcoRl /Mlul

PJ66633 6.3Kb

Bal I

Xhol

\

TEM beta-Lactamase

---

1 Leu Arg ti: CAG CTG CGTCAC

P:,’ G;: T:: CCA GAA ACG ---

PVUll

Fig. 1. Construction d(CCAGCTGG)

of plasmid

(New England

smaller MuI fragment

ofpPH

pJBS633.

(1) The BstEII

site of pTG2

was filled-in with T4 DNA polymerase

and the PvuII linker

BioIabs) was added by linker tailing (Lathe et al., 1984). (2) pBS129 was constructed

125 f Spratt et al., 1986), which carries the KmR (Km’) gene, with the corresponding

by replacing

fragment

the

from pBS 105

345

region for mature

/I-lactamase

origin was then joined from pJBS131

Construction and properties of translational fusions to TEM j?-lactamase

and the replication

to the StuI-PvuII

which contains

(c)

fragment

the KmR and TcR

genes and the fl phage on’ for ss DNA replication. The resulting

vector,

pJBS633

(6.3-kb),

KmR and TcR but does not provide to Ap since the mature /3-lactamase

If /?-lactamase

encodes

any resistance coding region is

is to be useful in protein

studies it must be sufficiently

robust

significant

activity when fused to

levels of enzymatic

N-terminal

portions

constructed

pJBS633 possesses a unique PvuII site at the start of the coding region for mature p-lactamase and

E. coli genes of known

fusions to TEM /?-lactamase

that it retains

of other proteins.

unexpressed.

translational

translational

fusions

We therefore

of six different

nucleotide

sequence

coding region of mature TEM /I-lactamase

can there-

fusion

determined

whether

proteins

DNA fragment is such that the ss plasmid DNA produced upon infection with phage fl provides a template that can be primed for sequencing from within the start of the coding region for mature B-lactamase and across the fusion junction.

therefore exhibited significant levels of p-lactamase activity. The sensitivity to Ap of individual cells of E. coli HB2 15 1 carrying each of the above pJB S633 derivatives was determined to ascertain whether it corre-

contains

that eliminate

strain, was cut partially

the &-acting

elements

for fl phage replication

were identified

as previously

A recombinant

(pJBS13 1) that contained

(Spratt

in the anticlockwise

direction

primer (Pharmacia).

Only the relevant

of the mature sequence Kb = kb.

the Hind111 and Smal

sites in the KmR gene (Spratt

with Stul and was ligated to the 71 I-bp Sspl fragment and morphogenesis

et al., 1986) and those with insertions the fl fragment

(but the clockwise restriction

form of TEM /I-lactamase

direction

in the correct on pJBS633)

(Spratt

is denoted

as judged

et al., 1986). pBS129

from pEMBL8

orientation

et al., 1986). Recombinants to provide

was identified

ss plasmid

by Ap’, and the nucleotide

sequence

across the Pm11 site are shown. His( + 1) is the first amino acid ofthe mature form ofTEM

DNA, prepared

containing

the fl origin

by restriction

mapping.

DNA that could be sequenced

by its ability to be sequenced

are shown. The presence

by their

+ (Dente et al., 1983) that

at the desired Shtl site were identified

sites in pTG2 and pJBS631

on pJBS633

activity

fusion

ability to protect E. coli against Ap in a ‘patch test’. In each case a DNA fragment was inserted into pJBS633 in such a way as to produce an in-frame fusion between the target gene and the p-lactamase coding region. Single-stranded plasmid DNA was then obtained from each of the recombinants and the nucleotide sequence across the fusion junction was verified. Derivatives of pJBS633 encoding fusion proteins that contained N-terminal regions of CAT, B-galactosidase, PBPlB, PBP3, PBPS and PBP6 were constructed (Table I). E. coli HB2151 containing each of the above pJBS633 derivatives grew when patched onto L agar plates containing 200 pg whereas strains HB2151 or Aplml HB2151[pJBS633] failed to grow. The more quantitative ‘spotting test’ showed that growth of the latter strains was inhibited by > 4 pg Ap/ml whereas each of the strains containing in-frame /?-lactamase fusions grew on at least 1 mg Ap/ml (Table I; high inoculum column). All of the above fusion proteins

from a dcm

/I-lactamase

the resulting

fore be obtained by inserting appropriate DNA fragments between the PvuII site and any of a number of unique restriction sites within, or just upstream from the TcR gene. Expression of fusion proteins from DNA fragments inserted between the MluI, EcoRI or Hind111 sites and the PvuII site requires the presence of a promoter on the inserted fragment. However, insertions using the EcoRV, BarnHI, or one of the other cloning sites within the coding region of the TcR gene will normally be expressed whether or not the incoming fragment contains a promoter as transcription will occur from the promoter of the TcR gene. In all cases transformants containing recombinant plasmids can be identified by the insertional inactivation of TcR and KmR is used as the selectable marker. The phage fl origin for ss DNA replication has been incorporated into pJBS633 so that the nucleotide sequence across the fusion junctions of any recombinants can be rapidly determined using dideoxy sequencing. The orientation of the phage fl

which has pomt mutations

retained

to the

and then

by pBR322 Hind111

of the unexpressed and corresponding

/I-lactamase.

coding region amino acid

Ap’ = ApR; Tc’ = TcR;

346

TABLE I Effect of inoculum size on the levels of ampicillin resistance conferred by r%lactamase fusion proteins a Plasmid carried by E. coli HB2151

Concentration of Ap (pg/ml) required to inhibit the growth of E. coli HB2151 derivatives spotted at: High inoculum (6 x lo6 cells spotted)

Low inoculum (40 cells spotted)

4 4

4 4

pJBS633-CAT-38/blaM pJBS633-&gal-1OjblaM pJBS633-PBPlB-27l/blaM pJBS633-PBP3-118~1~ pJBS633-PBP5-80/blaM pJBS633-PBP6-299/blaM

> 1000 > 1000 >lOOO > 1000 > 1000 > 1000

4 4 512 256 512 128

pJBS633-Tet-8/blaM pJBS633-Tet-27/blaM pJBS633-Tet-34/blaM pJBS633-Tet-97/blaM pJBS633-Tet-193~bl~ pJBS633-Tet-242~IaM pJBS633-Tet-97*/blaM pJBS633-Tet-97**/blaM

> 1000 > 1000 > 1000 1000 1000 > 1000 64 64

4 16 16 64 4 512 4 4

None pJBS633

pJBS633-Rop-9/blaM pJBS633-Rop-34/blaM pJBS633-Rop-43/blaM pJBS633-Rop-SO/bl~

512 1000 1000 1000

a The levels of Ap resistance of derivatives of E. cali HB2151 expressing fi-lactamase fusion proteins were measured at a high cell inoculum (the ‘spotting test’; MATERIALS AND METHODS, section c) and at a low inoculum (resistance of single cells; MATERIALS AND METHODS, section d). A standard nomenclature is used where, for example, pJBS633-CAT-38/blaM is a derivative of pJBS633 that expresses a fusion protein containing the N-terminal 38 residues of CAT fused to the mature form of TEM+lactamase. The derivatives pJBS633-Tet-97*/blaM and pJBS633-Tet-97**/blaM have fusions that join the fast or second nucleotides, respectively, of the 97th codon of the TcR gene to the PnrII site of pJBS633 and therefore are out-of-frame TcR gene-/I-lactamase fusions. The fusions of the rap and TcR genes to the coding region of mature TEM /I-lactamase were obtained from random fusion libraries constructed in pJBS633 (manuscript in prep~ation) and are expressed from their natural promoters. The fusions of CAT, &-galactosidase, PBPlB, PBP3, PBPS and PBP6 to #&lactamase were constructed by inserting convenient restriction fragments into pJBS633 using restriction sites in the corresponding genes which either produced blunt ends of the correct reading frame to be directly joined to the P&I site of pJBS633, or which gave sticky ends that could be converted to the correct reading frame by filling-in with T4 DNA polymerase. The PBP6 and p-galactosidase fusions were expressed from the uninduced luc promoter and the other four lesions from the promoter of the TcR gene. The numbering of the ammo acid sequences of PBPlB, PBP3 and PBP5 are those given by Broome-Smith et al. (1983; 1985) and Nakamura et al. (1983). The numbering of PBP6 is derived from the nucleotide sequence of the dacC gene (our unpublished results). The ‘&-galactosidase’ fusion, pJBS633-B-gal-lO/blaM, was obtained using the H&II site ofphage Ml3mp8 (Messing and Vieira, 1982) and therefore contained six residues from the N terminus of #&galactosidase, and four residues encoded by the multilinker of M13mp8, fused to #?-lactamase.

lated with the expected cellular location of the /J-lactamase moiety of each fusion protein, /Lgalactosidase and CAT are cytoplasmic proteins and the fi-lactamase fusion proteins derived from them failed to confer ApR on single cells of HB2151 (Table I; low inoculum column). PBPs 5 and 6 are syn-

thesised with signal peptides which direct the translocation of the proteins across the cytoplasmic membrane (Pratt et al., 1981). PBPlB and PBP3 are held in the membrane at their amino-termini with the bulk of the proteins translocated to the periplasm (Broome-Smi~ et al., 1985; A. Edelman, J.K. B.-S.

347

and B.G.S., in preparation). The fusion of p-lactamase to each of the above PBPs should therefore result in its translocation to the periplasm and, as expected, these fusion proteins did confer substantial levels of ApR resistance to single cells of HB215 1 (Table I; low inoculum column). The ability, or ~ab~ty, of each of the b-lactamase fusion proteins to protect single cells of HB2151 against Ap therefore correlated with the predicted cellular location of the j?-lactamase moiety. (d) Cellular location of the jklactamase moiety of fusion proteins The j.J-lactamase fusion proteins that protect single cells of E. coli against Ap should be those in which the j?-lactamase moiety has been translocated across the cytoplasmic membrane to the periplasm (Kadonaga and Knowles, 1985) and this was verified by showing that these fusion proteins were susceptible to protease digestion in spheroplasts. E. coli carrying derivatives of pJBS633 expressing each of the six fusion proteins were converted to spheroplasts and were incubated with proteinase K. After inhibition of the protease with PMSF, the proteins were fractionated by SDS-PAGE, blotted onto a nitrocellulose membrane, and probed with antibody to TEM @-lactamase. Fig. 2 shows that the PBPlBand PBP3-&lactamase fusion proteins were susceptible to protease digestion in spheroplasts (as were the PBPS- and PBP6-/&lactamase fusion proteins; not shown) whereas the CAT-j?-lactamase fusion protein (and the /?-galactosidase fusion protein; not shown) was not digested. The ability or inability of these fusion proteins to protect single cells of E. coli against Ap is therefore a reliable indicator of the translocation, or lack of translocation, of the p-lactamase moiety of fusion proteins across the cytoplasmic membrane. (e) Conditions for the selection of in-frame jIlactamase fusions from random fusion libraries One of the potential advantages of the vector pJBS633 is the ability to make a series of random fusions of a gene of interest to the coding region of mature /?-lactamase and to be able to select all of the in-frame fusions by the ‘patch test’ and then to predict the cellular location of the &lactamase

abc

d

ef

ghij

Fig. 2. Sensitivity offusion proteins to proteinase K digestion in spheroplasts. E. cob HB2151 derivatives were grown at 37°C in L broth to a density of 5 x 10’ bacteria/ml, and 20 ml aliquots were centrifuged (8000 x g for 3 min at room temperature), and the cell pellets were resuspended in 250 ~1 of 25 y0 sucrose; 30 mM Tris . HCl pH 8. The cells were converted to spheroplasts by the addition of 10 ~1 of 0.25 M EDTA, 10 ~1 of lysozyme (10 mg/ml) and 250 gl of distilled water. After 3 min at room temperature, > 95% of the bacteria had been converted into spheroplasts and 50 pl aliquots were incubated for 5 min at room temperature with or without proteinase K (Boehringer). Five pl of 100 mM PMSF were added and, after 3 min at room temperature, 50 ~1 of SDS gel solubiliser (Spratt, 1977) containing 20% (v/v) 2-mercaptoethanol was added and the samples were heated at 100°C for 3 min and fractionated by SDS-PAGE as described previously (Spratt, 1977). The proteins were transferred to a nitrocellulose sheet using a semi-dry blotter (Kyhse-Andersen, 1984) and were probed with a l/1000 dilution of a rabbit antiserum to TEM #?-lactamase using the Blotto system (Johnson et al., 1984). Horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) was used as the second antibody at a dilution of l/l000 and the blots were developed for 2 min with 0.4 mg/ml 3,3’-diaminobenzidine tetrahydrochloride and 0.012% hydrogen peroxide. Lane (a) HB215l[pTGZ] expressing wild-type TEM /?-lactamase; lanes (b)-(d) HB2151[pJBS633-CAT-38/blaM]; lanes (e)-(g) HB2151[pJBS633-PBP3-1 lS/blaM]; lanes (h)(j) HB215l[pJBS633-PBPlB-27l/blaM]. Proteinase K was added at a final concentration of 0 (lanes a,b,e and h), 200 @g/ml (lanes c,f, and i) or 1 mg/ml (lanes d,g and j). The positions of TEM /‘I-lactamase and the CAT-, PBP3- and PBPl B-b-lactamase fusion proteins are marked by the arrows in lanes (a), (b), (e) and(h), respectively. The PBPlB fusion protein exists in two forms which result from the presence of alternative translation starts in the ponB gene (Br~me-Smith et al., 198.5).

moiety of the fusion proteins on the basis of its ability, or inability, to provide single bacteria with resistance to Ap.

348

To examine the feasibility of using pJBS633 in this way we have chosen to study the TcR protein encoded by pBR322 as it is a typical example of an integral cytoplasmic membrane protein that consists of alternating long hybrophobic segments and shorter hydrophilic segments and which is believed to have a complex membr~e org~sation. A library of fusions of the coding region of mature p-lactamase to the TcR gene was obtained by cutting at random positions within the TcR gene using DNase I in the presence of manganese (Heffron et al., 1978) and joining the filled-in ends to the PvuII site of pJBS633 (details of the method for constructing random fusion libraries in pJBS633 and their use for the analysis of the topology of cytoplasmic membrane proteins will be published elsewhere). E. cob HB2 15 1 was transformed with DNA from the resulting ligation mixture and KmR, TcS, transformants were obtained. Those transformants containing inframe fusions were identified by patching colonies onto plates cont~ning 200 pg Ap/ml, and the level of protection against Ap afforded to single cells by the fusion proteins, and the nucleotide sequence across the fusion junctions were determined. Surprisingly, about 10% of the transformants that were believed to have in-frame fusions on the basis of the patch test were found to have out-of-frame fusions when the fusion junctions were sequenced. We presume that these transformants patched on Ap because a small amount of a fusion protein was expressed from these recombinants by frameshifting during translation. Since the inoculum achieved in the patch test is variable, we examined whether the more reproducible ‘spotting test’ could be used to discriminate ~~bi~ously between in-frame and out-of-frame fusions. A test in which 6 ~1 of overnight cultures of the transformants were spotted onto L agar plates containing 200 pg Ap/ml was found to be satisfactory as it allowed the growth of all of the transformants with in-frame fusions whereas all of those with out-of-frame fusions failed to grow. In particular it allowed the elimination of the class of out-of-frame fusions that appeared to produce some fusion protein by frameshifting (e.g., pJBS633-Tet97*/blaM) since these only grew on agar containing up to about 50 fig Ap/ml whereas all in-frame fusions grew on at least 250 pg Ap/ml (Table I ; high inoculum column). The properties of six typical in-frame fusions to

the TcR gene and four in-frame fusions from a random fusion library to the cytoplasmic product of the rop gene of pBR322 (Cesareni et al., 1982) are shown in Table I. Each of the fusions to the rap gene failed to protect single cells of HB2151 from killing by Ap, in agreement with the predicted cytoplasmic location of the fusion proteins. In contrast some of the fusions to the TcR gene provided single cells of HB2 151 with significant levels of protection against Ap whereas others gave no protection. The former class should be fusions of p-lactamase to positions in the TcR protein that are translocated to the periplasm whereas the latter class should be fusions to positions that are internal. The fact that some of the fusions to the TcR protein appear to result in translocation of fi-lactamase to the periplasm argues that this highly hydrophobic membrane protein contains active signals for translocation and does not passively partition into the membrane. The use of pJBS633 for the detailed analysis of the organisation of the TcR protein will be reported (J.K. B.-S., A. Edelman and M. Templin, in preparation). The vector can therefore be used to obtain all in-frame fusions of a target gene to the coding region of B-lactamase by using the patch test or spotting test, and the cellular location of the B-lactamase moiety can subsequently be determined by the Ap resistance level of single cells. Alte~atively, only those transformants that contain in-frame fusions which result in the translocation of &lactamase to the periplasm can be selected directly by plating cells that have been transformed with DNA from a ligation onto agar containing both Km and Ap.

(f) Conclusions

(1) pJBS633 provides a versatile vector for the construction of translational fusions to the coding region of mature TEM /$lactamase. A vector that allows the selection of translational fusions to mature TEM /3-lactamase by transposition has also been constructed (Tn-bluM; J.K. B.-S., unpublished). (2) The ability of ,&lactamase to be translocated across the cytoplasmic membrane when fused onto proteins that provide the appropriate export signals, and the ability to distinguish those fusions that result in translocation from those that do not, provides a convenient approach to the identi~cation of export

349

signals and the organisation of proteins in the cytoplasmic membrane. (3) The use of /I-lactamase fusions appears to have clear advantages over alkaline phosphatase fusion vectors (Manoil and Beckwith, 1985), as in the latter case only fusions that result in translocation of alkaline phosphatase across the cytoplasmic membrane are identified, whereas with pJBS633 all in-frame fusions can be identified. (4) The presence of the fl phage origin for ss DNA replication in pJBS633 allows the production of ss plasmid DNA and the rapid sequencing of the fusion junctions of recombinants.

tion. Proc. Natl. Acad. Johnson,

D.A., Gautsch,

Improved

technique

proteins

utilizing nonfat

J.R. and Elder, J.H.: dry milk for analysis

and nucleic acids transferred

Anal. Techn. Kadonaga,

A.E.,

Edge, M.D. and Knowles, sequence

to nitrocellulose.

of

Gene

1 (1984) 3-8.

J.T., Gautier,

Charles,

A.D.,

J.R.: Role ofthe /%lactamase

signal

in the secretion

Straus,

D.R.,

by Escherichiu coli. J.

of proteins

Biol. Chem. 259 (1984) 2149-2154. Kadonaga,

J.T. and Knowles,

for chemical

J.R.: A simple and efficient method

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