Mol Gen Genet (1992) 235:253-258

OIGG

© Springer-Verlag 1992

Lysis.protein T of bacteriophage T4 Meng-Ji Lu and Ulf Henning Max-Planck-Institut ffir Biologic, Corrensstrasse 38, W-7400 T/ibingen, FRG ReceivedApril 4, 1992 / Accepted June 14, 1992

Summary. Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage 2 is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of 2 (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18 000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme. Key words: Escherichia coli - Phage T4 - Lysis protein T

Introduction For cell lysis to occur at the end of the growth cycle of phage T4, expression of two phage genes, t and e, is required. Gene e (ge) codes for a lysozyme that degrades the murein layer of the cell envelope (Mukai et al. 1967). The function of the product of gene t (gt), protein T, is not well understood and the protein has, in fact, not even been identified. Amber mutants in gt, when grown in a sup ° host, are lysis defective (Josslin 1970, 1971); both phage and lysozyme are produced but the cellular metabolism continues and cell lysis does not Correspondence to: U. Henning

ensue. The latter defect can be overcome by artificially disrupting the cytoplasmic membrane. These observations suggest that protein T is responsible for changes in the membrane, allowing the lysozyme to reach its site of action. Genes t (or parts of them) of several Teven type phages have been cloned and sequenced (Riede 1987; Montag et al. 1987a, b). In the present work gt of T4 was used to identify protein T and to learn more about its mechanism of action.

Materials and methods Bacterial strains, growth conditions and phages. The

strains used are listed in Table 1. Cells were grown in L-broth or, for labelling with [35S]methionine, in M9 minimal medium (Miller 1972). Supplements were glucose (0.4%), glycerol (0.4%), ampicillin (50 gg/ml), kanamycin (30 gg/ml), isopropyl-/~-D-thiogalactopyranoside (IPTG, 1 mM) or bromo-chloro-indolyl-phosphate (40 gg/ml). The T4 amber mutants of gt, amtA3 and amtB5 (Josslin 1971) were kindly provided by W.B. Wood; 2ci857Sam7 and 2cI857Ram5 were gifts from N.E. Murray. Plasmids. In pT4t-2, gt is present on a 0.8 kb EcoRI fragment cloned into pUC19 (Montag etal. 1987a; pUC19: Yanisch-Perron et al. 1985); the reading frame is in the opposite orientation to the lac regulatory elements of the plasmid. It was restricted with EcoRI, religated, the ligation mixture transformed into strain M15F and the cells plated onto LB medium containing ampicillin and glucose. Replica plating onto this medium without glucose but containing IPTG identified colonies with gt in the other orientation, since they did not grow in the presence of the inducer; this plasmid was designated pT4t-1. A site for Sinai exists in gt at the codon for residue 34; the plasmid was opened with this enzyme and the resulting 0.6 kb fragment (second SmaI site in the vector) was ligated into pUCI8 (Yanisch-Perron et al. 1985), yielding pT4tA34 (Fig. 1). The construction

254 Table 1. Bacterial strains

Strain

Genotype

Source (reference)

MJ5F

A(lac-pro) ara rpsL recA

Drexler et al. (1986) MacIntyre et al. (1991) C. Manoil (Manoil and Beckwith 1985) C. Manoil (Manoil 1991) B. Mfiller-Hill

q580 lacZ AM15 F' lac~ ZAM15 pro + UH300 ompA

araD139 AlaeU169 rpsL relA thiA reeA proA or B ompA F'laclQZA M15 proA +,B +

CCJJ8

araD139 A (ara-leu) 7697 A lacX74 phoAA20 galE galK thi rpsE rpoB argEam recA1

CC202

same as CCl18 but carrying F'42 lacI3 zzf-2: :TnphoA thiA(lac-pro) F' lacl°Z +Y+

BMH61J

Strain UH300 was made ompA (not producing the OmpA protein) by selectionfor resistance to the OmpA-specificphage K3

SI1

153- ~ . - 15~. B po I b

ompA

SaS

r pEV 218

S B

IF ,o t p'T4tA34

I

pornpAt-1

I P

ompA

t

ompA

,~Sa, BAL31 153 • ,.ATC

eGG

~3 ATT

TCG

ATA

ATC""

Ire Arg lie Ser lie p

ii,

ompA

t

pompAt-2 Fig. 1. Construction of the ompA-t hybrid. Restriction site abbreviations: B, BamHI; Sa, SacI; S, SmaI. The numbers indicate amino acid positions. The residues beteen positions 153 of OmpA and 43 of T in the final product are encoded by vector DNA (thin lines) between the SacI and SmaI sites in pT4tA34; residues 34 (corresponding to the Sinai site in gt) to 43 were removed by the exonuclease treatment. The open box and the black box represent genes ompA and t respectively of plasmids pompAt-1 and pompAt-2 is presented in Fig. 1. In plasmid pEV218 (Freudl 1989) an ompA gene is present that carries a linker with multiple cloning sites between the codons for residues 153 and 154 of the corresponding protein. The 0.6 kb SacI-BamHI fragment from pT4tA34 was ligated into pEV218 digested with the same enzymes. In the resulting plasmid, pompAt-1, the fragment of gt is out of frame with respect to the ompA reading frame. It was opened with SacI, treated with exonuclease Bal31 and religated; screening of the resulting plasmids by D N A sequencing revealed many in-frame hybrid genes; one of these, ompAt-2, produced

a relatively stable fusion protein in large quantity. To remove the signal peptide-encoding region of ompA, pompAt-2 was cut with NruI plus HpaI and religated in the presence of the 8-mer BamHI linker C G G A T C C G ; this operation leads to a gene (present in pompAt-3) coding for an OmpA, which is missing most of the signal sequence and the first 45 residues of the mature protein (Freudl et al. 1985). Fusion of phoA to t was achieved by TnphoA mutagenesis (Manoil and Beckwith 1985). Plasmid pT4t-1 was placed into strain CC202; selection was on LB medium supplemented with ampicillin but without glucose and kanamycin, thus avoiding fusion genes which, when expressed in the absence of glucose, would already be lethal. Single colonies were streaked onto medium containing kanamycin (300 gg/ ml) and bromo-chloro-indolyl-phosphate. Plasmids from blue colonies were transformed into strain CCJ 18 using the same medium (kanamycin: 30 gg/ml) and those causing blue colour were analysed by D N A sequencing. In pT4tpho-J 1 the phoA sequence was found fused to the stop codon TAA of gt, changing this codon to TCT followed by the sequence present at the left end of TnphoA (Manoil and Beckwith 1985). Synthesis of this hybrid protein, induced with IPTG, was not lethal.

Antiserum and immunopreeipitation. Cells harbouring pompAt-3 were grown in LB medium containing ampicillin and IPTG. The fusion protein, which was produced in massive amounts and found in the insoluble fraction, was sedimented (20 rain, 50000 x g) together with the membranes and isolated by SDS-polyacrylamide gel electrophoresis followed by electroelution. A rabbit antiserum was obtained following a protocol described previously (Henning et al. 1979). Cells were labelled with [35S]methionine (50 I~Ci/ml, 1129 Ci/~tmol; New England Nuclear) as detailed below. Aliquots (109) cells were pelleted by centrifugation, suspended in 50 I.tl sample buffer (80 m M TRIS-HC1, pH 6.8, 2% SDS) and boiled for 5 rain; 300 ~1 buffer (50 mM TRIS-HC1, pH 8.0, 0.15 M NaC1, 2% Triton X-J00, 5 mM EDTA, 0.1% gelatin) was added and insoluble material removed by centrifugation. Five milligrams protein A-Sepharose

255 (Pharmacia), washed with the latter buffer, was added, the sample was shaken for 15 min at room temperature and the Sepharose removed by centrifugation. After adding 10 gl antiserum, the supernatant was incubated for 3 h at 0° C and 5 mg of protein A-sepharose was added, followed by a further 30 min shaking at 4° C. The Sepharose was washed three times with the same buffer and once with this buffer without Triton X-100. The Sepharose was finally suspended in 50 gl sample buffer, boiled for 5 min and the solubilized material analysed by SDS-polyacrylamide gel electrophoresis using 12.5% Laemmli-type gels (Laemmli 1970) and fluorography.

Cross-linking of proteins. For cross-linking, dithiobis succinimidylpropionate (DSP, Pierce) was used. Cells of strain M15F carrying pT4t-1 and pregrown in M9 medium containing ampicillin and glucose, were suspended (1 ml, 109 cells) in the same medium without glucose but containing glycerol, IPTG and [35S]methionine (concentration as given in the previous section). After 20 rain at 37° C the cells were washed with 20 mM sodium phosphate, pH 8.0, containing 20 mM NaC1 and resuspended with the same buffer to a concentration of 2 x 109/ml. DSP, dissolved in dimethylsulphoxide, was added at various concentrations and after 20 rain incubation at 20 ° C the reaction was stopped by adding glycine to a concentration of 0.1 M. The cells wer pelleted and processed for immunoprecipitation and electrophoretic analysis as described above, except that the boiling step was omitted. Molecular weight standard proteins were Rainbow Protein Molecular Weight Markers (Amersham). Other techniques. Preparation of cell envelopes and subsequent isopycnic sucrose density-gradient centrifugation were carried out as described by Osborn et al. (1972). For immunoblotting (Towbin et al. 1979), the antiserum and peroxidase-coupled goat anti-rabbit immunoglobulin (Miles) were used (Hawkes et al. 1982). For extraction with alkali (Davis et al. 1985), 1 ml cells were labelled with [35S]methionine and induced with IPTG as described above. One-half was used for immunoprecipitation and the other half was mixed with 2 ml 0.1 N NaOH. Cells were spun and proteins in the supernatant precipitated with trichloroacetic acid (final concentration, 10%), then washed with acetone, solubilized with sample buffer and used for immunoprecipitation as described above, fl-Galactosidase activity was measured with o-nitrophenyl-fl-D-galactoside as substrate (Miller 1972).

1.5 1.0

100

~0.8 0.6

>,> 50

7- o.4 0.2

0.0 0 20 4-0 60 80 100 120 14.0 rain

0 5 10 15 20 rain

Fig. 2. Phenotypiceffectsof expression of gt. For the measurements of turbidity (OD6oo) isopropyl-fl-D-thiogalactopyranoside(IPTG) was added at 20 rain (arrow).For determiningcolonyformingunits (survival), the inducer was added after taking the sample defining 100%. Strain M15F was used carryingeither pT4t-1 (I) or pUC19 (o)

(Fig. 2). This induction had a lethal effect: only about 20% of cells retained their colony-forming ability after 5 min of induction and only 2% survived after 15 min of induction (plated onto LB medium containing glucose). The induction did not cause lysis, i.e. no empty cells could be detected microscopically. We tested whether cytosolic proteins were released into the periplasm. After 30 min of induction periplasmic proteins were liberated by adding EDTA in the presence of 20% sucrose. The permeabilized cells were removed by centrifugation, proteins were precipitated with trichloroacetic acid and subjected to SDS-polyacrylamide gel electrophoresis. Staining of gels did not reveal any difference between the protein profiles derived from supernatants of induced cells or of cells treated identically but not carrying a plasmid. Clearly, while expression of gt was lethal, it did not cause any gross leakiness of the plasma membrane. In addition, when strain BMH611, harbouring pT4t-l, was grown for 30 min in M9 medium supplemented with glycerol and IPTG, high levels of fl-galactosidase were measured but no trace of this activity was found in the supernatant after pelleting the cells by centrifugation.

Complementation of T4gt and 2gS mutants Results

Phenotype of expression of the cloned gt In plasmid pT4t-1, gt was placed under the control of the lac regulatory elements. Growth of cells carrying this plasmid was normal in LB medium with added glucose, but stopped after about 20 min upon induction of expression of the gene by the addition of IPTG

It has been reported earlier that the cloned gt complements T4 gt amber mutants (Montag et al. 1987a). This complementation was quantitated. T4 gt amber mutants (tamA3 and tamB5) were tested on the sup° host MI5F harbouring T4t-1 grown in the presence of glucose. The efficiency of plating (e.o.p.) of both mutants was about 0.7 (revertants appeared at frequencies of 10-5-10-6). Using plasmid pT4t-2, which carries gt in an orientation opposite to that of pT4t-1 (and is thus promoterless),

256

1.0

/3

T-PhoA

36

OmpA

2~

T

"~ 0.5 -S.

0.2

0

I

I

I

I

I

15

30

4-5 min

60

75

Fig. 3. Complementation of 2ci857Sam7 by gt. Cells (strain M15F) lysogenic for this phage were thermoinduced at time 0, and either carried pT4t-1 (e, A) or no plasmid (o). Incubation after induction (10 min at 42° C) was in the presence of glucose (A) or of IPTG (o). Turbidity was measured at 600 nm

showed that recombinants appeared at a frequency of 10 -2. Hence, the low level of gt expression sufficed to complement the mutants efficiently. The functional equivalent of gt in phage 2 is gS, which is thought to form pores in the plasma membrane, allowing the murein-degrading product of gR to reach the periplasm (see the Discussion). Strain M15F was lysogenized with the thermoinducible 2ci857Sam7. When this lysogen carried pT4t-2, no lysis was observed for several hours after thermoinduction of the prophage. In the presence of pT4t-1 and in the absence of IPTG, cells began to lyse about 15 rain after thermoinduction and lysis also occurred when glucose was present, although at a slower rate (Fig. 3); in both cases phage progeny was liberated. When the medium contained 25% sucrose, the population was quantitatively converted to spheroplasts, i.e. the murein was degraded; lysis of the spheroplasts at low osmotic pressure also released 2 progeny. As a control, strain M15F carrying pT4t-1 was also lysogenized with 2cI857Ram5 which cannot express gR. Thermoinduction of this prophage did not cause any lysis under the conditions that lead to lysis using 2ci857Sam7. Clearly, T4gt could replace 2gS.

Identification of protein T It was not possible to find a stainable candidate for protein T on electrophoretograms of cells harbouring pT4t-1 and induced with IPTG. Therefore, a gene was constructed in which part of the ompA gene (encoding the outer membrane protein OmpA) is fused to a fragment of gt. This gene codes for OmpA from residues 44 to 153 (i.e. lacks the signal peptide) fused to residue 35 of protein T (Fig. 1 ; see the Materials and methods). In addition, TnphoA mutagenesis produced a hybrid with the gene for the periplasmic alkaline phophatase (PhoA) fused to the stop codon TAA of gt. This codon

1

2

3

Fig, 4. Identification of T protein. An autoradiogram of an electrophoretogram (12.5% acrylamide) of radioactively labelled immunoprecipitates is shown. Antiserum against the OmpA-T fusion protein was used. Lane 1, strain UH300 (ompA) carrying pT4t-1; lane 2, the same strain harbouring pompAt-2; lane 3, the ompA + parent of strain UH300 without plasmid. All three cultures were grown for 30 rain in the presence of IPTG and [35S]methionine. The faint band in lane 2 just above protein T most probably represents a degradation product of the hybrid polypeptide. Numbers on the left margin represent molecular weights in kDa

was changed to TCT and was followed by the phosphatase gene not encoding the signal sequence (see the Materials and methods). Both hybrids were under the control of the lac regulatory elements and induction caused synthesis of both proteins without toxic effects. An antiserum, which was raised against the OmpA-T fusion protein, did not, of course, react with PhoA but recognized the OmpA protein and the T-PhoA hybrid (Fig. 4); hence, it contained antibodies against T. For identification of T, cells of strain UH300 (not producing the OmpA protein) carrying pT4t-1 were labelled with [aSS]methionine and whole cell lysates were used for immunoprecipitation. A protein of molecular mass ,-~25000 was precipitated (Fig. 4), but only when labelling was performed in the presence of IPTG; labelling in medium containing glucose or of cells harbouring the parental plasmid pUCI9 (IPTG present) did not cause the appearance of this polypeptide. Unquestionably, therefore, the labelled protein represents T. Separation of outer and inner membranes, derived from cells induced with IPTG, by isopyknic sucrose density-gradient centrifugation (Osborn et al. 1972) showed the protein to be associated with the plasma membrane (not shown). Proteins anchored in a membrane via a hydrophobic anchor are generally insoluble in alkali (Davis and Model 1985; Davis et al. 1985; Sakaguchi et al. 1987). Protein T in cells harbouring pT4t-1 and induced with IPTG for 30 rain proved to be entirely insoluble in 0.1 N NaOH (see the Materials and methods). Hence and expectedly, we are dealing with an intrinsic plasma membrane protein. The identification of protein T allowed investigation of possible interactions between protein T and other proteins. To cells carrying pT4t-l, IPTG and [35S]methionine were added; after 20 min the culture was treated

257

200

92.5 69

m&. 6

--30

-1

2

3

21.5

Fig. 5. Autoradiographic analysis of cross-linkingexperiments. The concentrations of the cross-linker dithiobis succinimidylpropionate (DSP) were: lane 2, 0.05 mM; lane 3, 0.1 raM; and lane 4, 0.2 mM. The sample in lane 1 was processed without DSP. When samples such as those applied to lanes 2-4 were processed for electrophoresisin the presence of fl-mercaptoethanol, only the monomer was visible. The positions of the molecularweight standard proteins are given in kDa

4.

with dithiobis succinimidylpropionate at various concentrations. An electrophoretic analysis of the immunoprecipitated proteins is shown in Fig. 5. In the absence of the cross-linker only protein T was visible. The major products consisted of a group of polypeptides with apparent molecular weights between 46 and 50 kDa, most likely dimers of T. Since the protein contains 13 lysine residues, a number of conformationally different dimers may be possible, exhibiting slightly different electrophoretic mobilities. With increasing concentrations of the cross-linker, oligomers of slower mobilities became more prominent; those corresponding to molecular weights of 77 and 100 kDa may represent trimers and tetramers, respectively. These results have to be considered in light of the fact that the concentration of T, even after induction with IPTG, must still be very low. It was not possible to detect the protein by immunoblotting of lysates of whole cells, and visualization of the polypeptide with this method could only be achieved when the membrane fraction of induced cells was used. Therefore, the formation of oligomers cannot be accidental, i.e. cannot possibly be caused by the existence of a dense layer of monomers. We conclude that protein T acts in an oligomeric form.

Discussion

Protein S of phage 2 is the functional equivalent of protein T. S increases the permeability of the plasma membrane (Wilson 1982), most probably allowing protein R, a transglycosylase (Bienkowska-Szewczyk etal. 1981), to reach the murein layer (Garrett and Young 1982; Reader and Siminovitch 1971). It has been shown that the cloned genes encoding protein 19 (a mureindegrading enzyme) of phage P22, and 2gR, can complement both 2gR- and P22g19- mutants. The equivalent of S in P22 is protein 13, which has 89% sequence simi-

larity to S. For 2R, however, no such similarity to protein 19 could be found (Rennell and Poteete 1985). Hence, in this case, S of 2 can functionally substitute for the unrelated protein 19 of P22. A comparable situation is represented by the complementation of a defective 2gS by gt of T4. The 218-residue T is about twice the size of the 107-residue protein S (Montag et al. 1987a; Sanger et al. 1982) and the two polypeptides show no significant sequence similarity. Also, T4 lysozyme does not share sequence similarity with the transglycosylase of 2. Despite their different primary structures the membrane topologies of S and T are probably very similar. S exists, associated with the plasma membrane, as a homooligomer (Altman et al. 1985; Zagotta and Wilson 1990). In view of the interchangeability of S and T it was therefore not surprising to find that T is also an intrinsic membrane protein of homooligomeric nature. It has been suggested that S crosses the plasma membrane by means of three lipophilic c~-helices (Raab et al. 1986). In T, a stretch of 15 lipophilic residues exists which, however, is not very likely to serve as a membrane anchor. Such anchors are normally longer (yon Heijne 1985) and it has been shown that stable membrane integration can be achieved with 16 or more lipophilic residues but not with 10-14 (Davis and Model 1985; Davis et al. 1985). There are no other reasonable candidates for potential membrane anchors. Evidene has been obtained that proteins may be anchored in the plasma membrane via an 18-residue amphiphilic c~-helix (Jackson and Pratt 1987, 1988). A good candidate for such a helix in S exists in the sequence starting at position 5: -Gln-Lys-His-Asp-Leu-Leu-AlaAla-Ile-Ile-Ala-Ala-Lys-Gln-Gln-Gly-Ile-Gly-. Two such candidates are discernible in T, one involving the 15 lipophilic residues mentioned above (starting at position 35) and another, starting with residue 181: -AspAsn-Ile-Tyr-Ala-Gly-Thr-Ile-Thre-Met-Tyr-Trp-TyrArg-Asn-Asp-His-Ile-. (These helices are evident when the sequences are written as helical wheels accoring to Schiffer and Edmundson (1967).) The existence of an amphiphilic e-helix could easily explain how the polypeptides could be arranged in the membrane as oligomers. An oligomer consisting of amphiphilic e-helices would also be well suited to form a pore allowing 2R or T4 lysozyme access to the murein. All data taken together suggest that the lysis proteins form pores. For the following reasons we suggest that these (hypothetical) pores possess specificity for the murein-degrading enzymes. Enough protein T to complement T4 gt or ;~gS mutants was expressed from the cloned gene, under the control of the lac regulatory elements, even in the presence of glucose, a condition under which T is not even detectable immunologically and certainly not more than 100 copies (and probably considerably less) are present per cell. This, together with the absence of detectable leakage of other cytosolic proteins strongly suggests, of course, that there is specificity for the binding of the hydrolases to S or T. The three hydrolases discussed have two common denominators. Firstly, they are all of about the same size (T4 lysozyme, 18400, Tsugita etal. 1968; 2 transglycosylase, 17500, Bienkowska-

258 Szewczyk a n d T a y l o r 1981; p r o t e i n 19 o f P22, 16000, Rennell a n d Poteete 1985). Secondly, t h e y are very basic with a l m o s t identical p r o p o r t i o n s o f acidic a n d basic residues: T4 l y s o z y m e , 18 acidic plus 26 b a s i c ; 2 transglycosylase, 20 acidic plus 24 basic; p r o t e i n 19, 16 acidic plus 21 basic. This w o u l d b r i n g their isoelectric p o i n t s to b e t w e e n p H 10 a n d 11. A n u m b e r o f o t h e r cytosolic p r o t e i n s o f this size r a n g e ( a n d o f still l o w e r m o l e c u l a r weights) exist; a p p a r e n t l y none, however, are o f such basic n a t u r e (Philips et al. 1987). Thus, the specificity c o u l d be c a u s e d b y this c h a r a c t e r a n d , in a d d i t i o n , there c o u l d be a size l i m i t a t i o n . These features w o u l d p r e d i c t a p e r m a n e n t l y o p e n p o r e with specificity for a defined class o f ligands, j u s t as for e x a m p l e , the o u t e r m e m b r a n e m a l t o p o r i n has specificity for m a l t o d e x t r i n s , w h i c h is n o t s h a r e d b y o t h e r p o r i n s ( W a n d e r s m a n et al. 1979). Acknowledgements. We thank C. Manoil, B. Mfiller-Hi11, N. Mur-

ray and W.B. Wood for bacterial strains and phages and R. Freudl for plasmid pEV218.

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