Arch Microbiol (1992) 158:235-248

Archives of

Hicrobiology

9 Springer-Verlag1992

Minireview

Molecular interaction between bacteriophage and the gram-negative cell envelope Knut J. Heller Universit/it Konstanz, Fakult/it fiir Biologie, Universit/itsstrasse 10, Postfach 5560, W-7750 Konstanz, Federal Republic of Germany Received March 16, 1992/AcceptedMay 8, 1992

Bacteriophages recognize their host bacteria through highly specific binding to receptors located on the bacterial cell surface. During the last decades geneticists have taken advantage of this fact and have used bateriophages as selective agents to identifiy and characterize components of the bacterial cell surface. A number of outer membrane proteins of gram-negative bacteria have been identified this way, e.g. TonA (T-one), Bfe (BF23 and E-colicins), Lamb (lambda) and Tsx (T-six). Probably every structure exposed on the cell surface may serve as a phage receptor. The involvement of flagella, pill, capsules, teichoic acid, tipopolysaccharide, and protein as phage receptors has been discussed in several reviews (Ackermann and DuBow 1987; Beumer et al. 1984; Braun and Hantke 1977; Konisky 1979; Lindberg 1973, 1977; Osborne and Wu 1980; Reeves 1979; Schwartz 1980). In recent years the structure and function of both outer membrane and phage proteins has been intensively studied. The studies gave insight into domains of receptor proteins exposed to the outer surface of bacteria and domains of phage proteins directly participating in binding to the target sequences on the receptors. In this review I will mainly focus on the molecular structures that govern the interactions between phage proteins and proteins of the outer membrane of gramnegative bacteria. In the first and second part I will summarize the current knowledge about receptor and phage proteins and their domains involved in phage/ receptor recognition. In the third part I will try to analyze the molecular mechanisms of receptor recognition by T-even-type phages. Finally, I will briefly discuss the mechansim of phage D N A penetration through the bacterial cell envelope.

Receptor proteins Newly developed molecular biology techniques have helped elucidate the membrane topology of several outer

Abbreviation: aa, amino acid(s)

membrane proteins of the gram-negative bacterium Escherichia coli in recent years. One important approach used has been the analysis of phage resistant mutants which synthesize an altered receptor protein. Regions of the receptor protein identified this way are considered to face the outer medium. However, this conclusion can be drawn only when the mutations are assumed to have purely local effects and not alter the overall structure or folding of the entire or a considerable portion of the protein. To exclude the latter possibilities it is necessary to demonstrate that the mutant protein still shares common properties with the wild-type protein, such as synthesis in equal amounts, localization in the outer membrane, equal stablity, and equal resistance to proteases. The strongest argument against a grossly altered structure of the receptor protein is retention of most pysiological functions of the wild-type protein. However, considering overlapping functional domains, this may not always be possible. Outer membrane proteins with published amino acid (aa) sequences are listed in Table 1. The number of aa of the mature proteins have been determined experimentally by N-terminal aa sequencing in only few cases; usually, the cleavage site of leader peptidase has been proposed by comparison with consensus sequences for signal peptide and cleavage site structures (von Heijne 1987).

BtuB, Bfe, FhuA, SidK Outer membrane proteins involved in uptake of ferric iron and vitamin B12 are components of complex, high affinity transport systems. The unique feature of these systems is that they require an energized cytoplasmic membrane and the TonB protein for translocation of their substrates across the outer membrane and/or release into the periplasmic space. TonB is a periplasmic protein, anchored within the cytoplasmic membrane by an Nterminal anchor sequence and directly interacting with the outer membrane receptors. Through this interaction TonB energetically couples the cytoplasmic membrane to active transport processes across the outer membrane (for a recent review see Postle 1990).

236 Table 1. Receptor proteins in the outer membrane of enterobacteria"

Protein b

Size c

Physiological function

Coliphage d

FhuA 1 BtuB 2 FadL 3 LamB 4 LamBs.s. 5 LamBs.t. 6 OmpC v

714 594 421 421 421 427 346

ferrichrome receptor vitamin B12 receptor fatty acid uptake maltodextrin-specific pore maltodextrin-specific pore maltodextrin-specific pore general diffusion pore,

OmpCs ~ 8 NmpC 9 Lc 9 OmpF lo PhoE 11

356 342 342 340 330

PhoEE.a. 12

329

PhoEK p 12

329

OmpA 13

325

OmpAs d 14

330

OmpAs.t. is

329

OmpA~., 16 OmpAs m 17 Tsx is Tsxs.t.19 TSXE.a.19 TSXK.p.19

329 338 262 255 262 262

general diffusion pore general diffusion pore general diffusion pore general diffusion pore, general diffusion pore, anion-selective general diffusion pore, anion-selective general diffusion pore, anion-selective F'-mediated conjugation, structural function F'-mediated conjugation d, structural function F'-mediated conjugation d, structural function structural function structural function nucleoside-specific pore nucleoside-specific pore nucleoside-specific pore nucleoslde-specific pore

T1, T5, phi80, UC-1 BF23 T2 lambda, K10, TP1, SS1 lambda TuIb, T4, SS1, TP2, Me1, PA-2, HK253hrk ? ? SQ108h2 Tula, TP1, TP2, (T2) SQ 108 TC45 TC45 TC45 Ox2, K3, TuII*, MI Ox2 Ox2 Ox2 r Ox2 r T6, Oxl, K9, H8 T6, Oxl, Kg, H8 p T6, Oxl, K9, H8

a Proteins with known amino acid (aa) sequence are listed only. References for aa sequences are: 1Coulton et at. 1986; 2Heller and Kadner 1985; 3Black 1991; 4Clement and Hofnung 1981 ; SRoessner and Ihler 1987; 6Franzoc et al. 1990; 7Mizuno et al. 1983 ; 8Venegas et al. 1988; 9Blasband et al. 1986; l~ et al. 1982; 11Overbeeke et al. 1983; 12van der Ley et al. 1987; 13Beck and Bremer 1980; 14Braun and Cole 1982; 15Freudl and Cole 1983; 16Braun and Cole 1983; 17Braun and Cole 1984; lSBremer et al. 1990; 19Nieweg and Bremer (unpublished results) b Indices indicate bacterial strains. E.a., Enterobacter aerogenes; S.t., Salmonella typhimurmm: S.y., Salmonella typhi; K.p., Klebsiella pneumomae: S.d., Shigella dysenteriae, S s., Shigella sonnei ; S.m., Serratia marcescens. No indices, Escherichia coli ~ No. of aa residues of mature protein d For references of host-specificities see Beumer et al. (1984); Phages SQ108 and SQ108h2 have been described by Fralick et al. (1990). Receptor proteins from different species were tested in E. coli. ?, no data; - , resistent against all coliphages using the E. coli protein as receptor; r, resistent; P, partially resistent

A m o n g the T o n B - d e p e n d e n t r e c e p t o r p r o t e i n s o n l y B t u B a n d F h u A (formerly T o n A ) o f E. coli a n d Bfe a n d S i d K o f S a l m o n e l l a t y p h i m u r i u m , i n v o l v e d in v i t a m i n B ~2 (BtuB, Bfe) a n d F e 3 + - f e r r i c h r o m e ( F h u A , S i d K ) u p t a k e , h a v e b e e n r e p o r t e d to serve as p h a g e r e c e p t o r s (for reviews see B e u m e r et al. 1984; L u g t e n b e r g a n d v a n A l p h e n 1983). F h u A - s p e c i f i c p h a g e s are T1, T5, phi80, a n d U C - 1 ; ES18 is a S i d K - s p e c i f i c p h a g e . BtuB a n d Bfe a r e b o t h r e c e p t o r s f o r BF23. T1 a n d phi80 r e q u i r e the t o n B e n c o d e d p r o t e i n for irreversible b i n d i n g to F h u A ; a d s o r p t i o n to F h u A is reversible o n l y in t o n B cells o r w i t h the i s o l a t e d p r o t e i n ( H a n c o c k a n d B r a u n , 1976, H o f f m a n n et al. 1986). This is p r o b a b l y d u e to a different c o n f o r m a t i o n o f F h u A i n d u c e d b y i n t e r a c t i o n with T o n B . So far m u t a n t s specifically affecting the p h a g e r e c e p t o r p r o p e r t i e s o f BtuB a n d F h u A h a v e n o t been described, instead pleiotropic mutants simultaneously

affecting all r e c e p t o r a n d t r a n s p o r t f u n c t i o n s h a v e been f o u n d ( H a n t k e a n d B r a u n 1978; G u d m u n d s d o t t i r et al. 1988; C a r m e l et al. 1990, Schultz et al. 1989). Mutants resistant to phage T5 but able to grow on ferrichrome as sole iron-source have been isolated (Sommer and Heller, unpublished data). FhuA was not detectable in these mutants even by the Western blot technique. An effect on transcription and/or translation initiation can be ruled out, since the corresponding DNA-regions have been sequenced and shown to be unaltered (Sommer and Heller, unpublished data). A possible explanation could be that very unstable proteins are synthesized. FadL

F a d L f u n c t i o n s as a f a t t y acid-specific t r a n s p o r t p r o t e i n in the o u t e r m e m b r a n e o f E . coli (Black 1988, 1990, 1991 ; K u m a r a n d Black, 1991). I n a d d i t i o n it serves as the m a j o r r e c e p t o r for p h a g e T2 (Black 1990).

237 Originally, O m p F had been reported to constitute the m a j o r receptor for T2 (Hantke 1978). However, due to the low frequency of spontaneous mutations resulting in T2-resistance (Hantke 1978) a two-step mutational process has been proposed (Lenski 1984). This was confirmed by M o r o n a and Henning (1986) who identified a new locus, ttr, that appeared to encode a receptor protein for phage T2. Only ttr ompFdouble mutants were totally resistant to T2, whereas ompF mutants were not at all and ttr mutants were only slightly affected in their susceptibility to T2 ( M o r o n a and Henning 1986). Finally, Black (1988) showed that ttr and f a d L were identical genes. F a d L is synthesized as a precursor protein of 448 aa residues with a cleavable signal sequence of 27 aa residues (Black 1991). In contrast to other outer m e m b r a n e proteins F a d L contains an abundance of hydrophobic aa (Black 1991). This m a y reflect its function as a channel, specific for hydrophobic substrates like the long-chain fatty acids (Black 1990). However, the nature of transport is not clear. So far, data on the membrane topology of FadL have not been published nor have mutants been described which are specifically affected in their T2 receptor function. The only mutants characterized on a molecular level have been obtained by linker insertion mutagenesas at five different sites within thefadL gene (Kumar and Black 1991). The mutant proteins, which each have suffered insertions of two amino acids, show defects in oleate binding or transport (Kumar and Black 1991). At least, the insertions define sites not directly involved in reception by phage T2.

LamB L a m B is a channel of the outer m e m b r a n e of E. coli specific for maltose and maltodextrins (Szmelcman and H o f n u n g 1975). A 2D-model of L a m B folding in the outer mebrane has been proposed, which is based on the following approaches to analyse the L a m B topology: i) analyses of mutations affecting binding of phages lambda and K10; ii) analyses of mutants impaired in maltodextrin transport; iii) analyses of mutations affecting recognition of monoclonal antibodies; iv) identification of protease-sensitive peptide bonds; v) analyses of antibody-binding to inserted foreign epitopes (Charbit et al. 1988 and references therein; Charbit et al. 1991). According to the model L a m B spans the outer m e m b r a n e 16 times, forming eight loops exposed to the surrounding medium. Mutations affecting binding of bacteriophages are predominantly found in the C-terminal two-thirds o f L a m B : they are located in loops 4, 5, 6, and 8 (Boulain et al. 1986; Charbit et al. 1988; 1991; Heine et al. 1988; Gehring et al. 1987). Coincidently, loops 4, 5, 6, and 8 form four of the five variable regions, when E. coh LamB is compared to LamB of S. typhimurium (Francoz et al. 1990). The latter, when expressed in E. coli, is resistant to the E. eoli LamB-specific phages K10 and lambda, including the host-range mutants lambdah ~ (one-step mutant, able to infect the lambda-nonadsorbing strain CR63) and lambdahh* (two-step mutant, able to anfect pop 1067 in addition to CR63) (Appleyard et al. 1956; Hofnung et al. 1976). Two lamB mutations affecting loop 1 have been descrabed as causing lambda resistance (Gehring et al. 1987; Heine et al. 1988). The corresponding region of LamB is critical for membrane insertion and trimer formation;

therefore, indirect effects on phage adsorption may be possible (Heine et al. 1988). The fact that the mutants are sensitive to lambdah ~ and lambdahh* (Gehring et al. 1987) does not exclude redirect effects, since both host-range mutants most likely belong to the "trigger-happy"-type (see chapter on receptor recognition). Most of the lamB point mutations analyzed so far cause resistance to lambda but not to the host range mutants lambdah ~ and lambdahh*. Only two exceptions exist: the G151D (change from glycine to aspartic acid at aa position 151 of mature LamB) mutation causes resistance to lambda and lambdah o, and the G401D mutation causes resistance to lambda and partial resistance to lambdah ~ Both are sensitive to lambdahh* (C16ment et al. 1983). Interestingly, the G401V mutation causes only partial resistance to lambda and no resistance to lambdah ~ (Gehring et al. 1987). The observed cross-resistance for lambda and K10 of most of the LamB mutants argues for overlapping receptor sites. However, the occurance of other mutants demonstrates that the receptor sites are not identical. The K10-resistant mutants S154F and F155S within loop 4 are sensiUve to lambda (Roa and C16ment 1980; Charbit et al. 1984). On the other hand, the lambda resistant mutants $250F of loop 6 and G382D and G401D of loop 8 are sensitive to K10 (Charbit et al. 1984; Gehring et al. 1987). The S152F mutation causes lambda resistance while the S152C mutation does not; only after further modification of the cysteine does lambda adsorption become inhibited. The same is true for the S153C and S154C mutations (Francis et al. 1991). The results indicate that aa at positions 152, 153, and 154 are not involved in the actual binding of phage lambda. Mutations or modifications affecting these aa most likely indirectly interfere with binding of lambda either by changing the local secondary structure of loop 4 of LamB or by sterical interference through bulky aa side chains (Francis et al. 1991). Spontaneous ejection of D N A from wild-type lambda, triggered by binding to purified E. coli LamB, only occurs in the presence of chloroform or alcohol (RandallHazelbauer and Schwartz 1973). However, the solvents are no longer needed if the extended host range mutants h ~ and hh* are used or if purified L a m B from ShigeIla sonnei 3070 instead of L a m B from E. coli is used (Roessner and Ihler 1987). The S. sonnei L a m B differs from the E. coli L a m B in a single region: of the ten aa residues 381 to 390 within loop 8, seven are different (Roessner and Ihler 1987). Within this region of E. coli L a m B mutations have been described which cause l a m b d a resistance (C16ment et al. 1983; D e s a y m a r d et al. 1986). The G401D mutation (lamB110), which prevents ejection of lambda D N A although binding still occurs (Braun-Breton and Hofnung 1981; Roessner and Ihler 1987), appears to be involved in D N A ejection in addition to residues 381 to 390.

OmpC, OmpF, PhoE, NmpC, Lc The outer m e m b r a n e proteins O m p C , O m p F , PhoE, N m p C , and Lc f o r m a family of closely related general diffusion pores, termed porins (Jeanteur et al. 1991). Due to the lack of substrate binding sites they are not specific for certain substrates, however, as a consequence of the pore properties they exibit selectivity for molecules with respect to size, charge, and hydrophilicity of these molecules (for reviews, see Benz and Bauer 1988; Nikaido 1992). While O m p C , O m p F , and PhoE are encoded by c h r o m o s o m a l genes o f bacteria, Lc and N m p C are encoded by genes located on the c h r o m o s o m e of the lambdoid, temperature phage PA-2 and of a defective

238 p r o p h a g e , respectively (Blasband et al. 1986). Expression o f the Lc pore during P A - 2 lysogeny leads to a substantial reduction o f O m p C and O m p F expression (Fralick and Diedrich 1982). A similar kind o f lysogenic conversion has been observed with temperate phage H K 2 5 3 h r k ( V e r h o e f et al. 1987). T h e m e m b r a n e t o p o l o g y seems to be identical for all of the porins, i.e., they all are predicted to c o n t a i n 16 membrane-spanning/?-sheets, with the N - a n d C-terminal ends facing the periplasm. The periplasmic loops are very small (1 to 8 aa residues) and m u c h less p o l a r t h a n the relatively large loops facing the external m e d i u m (Jeanteur et al. 1991 ; T o m m a s s e n 1988). Similar characteristics have been predicted for the N-terminal h a l f o f O m p A which spans the outer m e m b r a n e eight times (Vogel a n d J/ihnig 1986). The recently published threedimensional X - r a y structure o f the porin f r o m Rhodobacter capsulatus (Weiss et al. 1991) confirms the prediction (Jeanteur et al. 1991). The loops facing the external m e d i u m show the highest degreed o f variation between the different porins (Jeanteur et al. 1991). The few p o i n t m u t a t i o n s resulting in p h a g e resistance are located within these loops: G 1 6 9 C o f O m p C , resistant to T u I b but sensitive to T4 ( M o n t a g et al. 1990); G62, G154, and L250 o f O m p C (Misra and Benson 1988); R 1 5 8 H o f P h o E , resistant to T C 4 5 but sensitive to the host-range m u t a n t T C 4 5 h r N 3 ( K o r t e l a n d et al. 1985). The involvement of the OmpC region around G169 in TuIb receptor function has been confirmed by the use of proteinase K which cleaves OmpC in this region. Treatment of ceils with proteinase K completely abolished the ability of OmpC to act as a receptor for TuIb but did not affect the receptor activity for Ox2hl0 (Morona et al. 1985b). Regions of PhoE required for TC45 adsorption were further identified by two different approaches, the construction of small deletions within PhoE (Agterberg et al. 1989) and the construction of OmpC'-'PhoE (Tommassen et al. 1985) and of PhoE'-'OmpC chimeric proteins (Van der Ley et al. 1987b). Deletion of seven aa residues (158-164) including R158 resulted in TC45 resistance, whereas deletion of twelve aa residues (195-206) did not affect TC45 sensitivity of the cells (Agterberg et al. 1989). The studies with chimeric proteins supported the deletion analyses, aa residues of PhoE C-terminal to residue 165 could be replaced by OmpC sequences without loss of TC45 receptor specificity (Van der Ley et al. 1987b). With the reverse chimeric protein it was shown that the N-terminal 52aa's of PhoE could be replaced by the corresponding parts of OmpC without destroying the receptor function for TC45 (Tommassen et al. 1985). The dependency of TC45hrN3, the host range phage isolated for the R158H mutant of PhoE, appears to be partly relieved for the corresponding externally exposed region around R158 (Tommassen et al. 1985). Taken together, the results indicate that of the surface-exposed regions of PhoE, termed a through h, three - b, c, and d - are involved in the formation of the receptor area for TC45 (Van der Ley et al. 1987b). However, comparison of the aa sequences of PhoNs from E. coli, Enterobacter cloacae, and Klebsiella pneumoniae, which are all recognized by phage TC45 (Van der Ley et al. 1987a), shows that only region c is absolutely conserved, while regions b and d differ more or less from each other. ObviousIy, these changes are not critical for TC45 binding. The data with chimeric proteins have to be interpreted with caution, since an external loop of one protein may be functionally replaced by a corresponding loop of the other protein, especially if the loops share high degrees of homologies. With the same reservations, surfaced-exposed regions of OmpC

functioning in phage reception were identified (Van der Ley et al. 1987b). These regions were: e and/or f for phage Me 1; c, d, e and/or f for TuIb, which is consistint with the finding that cleavage by proteinase K within region d destroys the TuIb receptor function (Morona et al. 1985b); c and/or d and e and/or f and h for PA-2; c and/or d and e and/or f for SS4; a and/or b for HK253hrk.

OmpA The O m p A protein is required for the structural integrity o f the outer m e m b r a n e and for F ' - m e d i a t e d conjugation. It also appears to function as an inefficient general diffusion pore ( S u g a w a r a and N i k a i d o 1992). Besides these functions it serves as a receptor for phages K3, Ox2, and TulI*. The p o r t i o n o f O m p A involved in phage reception is confined to the N-terminal half o f the protein; the C-terminal h a l f is located in the periplasm (Bremer et al. 1982). As shown in Fig. 1 the N-terminal part traverses the m e m b r a n e eight times, thereby f o r m i n g four surface-exposed loops a r o u n d aa residues 25 (loop 1), 70 (loop 2), 110 (loop 3), and 154 (loop 4), respectively ( M o r o n a et al. 1984; Freudl et al. 1986; Vogel and J/ihnig 1986). Phage-resistant m u t a n t s have been isolated and their phenotypes have been characterized (Cole et al. 1983; M a n o i l 1983). The resistance patterns indicate that the binding sites for different ligands are distinct but overlapping (Manoil 1983). All four loops m a y be involved in the f o r m a t i o n o f the binding site for a single phage. This is certainly true for Ox4, Ox5 and may be true for Ox2. Other phages like TulI*-6 and K3 may only require loops 1,2, and 3 or loops 2 and 3, respectively (Morona et al. 1984; Morona et al. 1985a). However, although a relatively high number of phageresistant mutants have been analyzed and several mutants have been independently isolated several times (Morona et al. 1985a), the possibility still exists that TuII*-6 and K3 may require as their receptor all four loops of OmpA. This could be the case if the altered aa of a loop in a mutant protein were not directly involved in the formation of the phage-binding site. As has also been seen with LamB, the nature of an aa substitution may be critical. For example, in OmpA the substitution G(70)V results in resistance to K3, while substitution G(70)C does not affect K3 binding (Morona et al. 1985a). Mutants within region "70" show an LPS-dependent phage resistance pattern that may be caused by a conformational change of the polypeptide chain induced by interaction with core sugars of the LPS (Morona et al. 1984). The data with phage-resistant mutants are complemented by sequence data of OmpA proteins from different enterobacterial species (Beck and Bremer 1980; Mowa et al. 1980; Braun and Cole 1982, 1983, 1984; Freudl and Cole 1983). Within the N-terminal half the highest variation among the five different OmpA proteins occurs in loops around aa residues 25, 70, and 110 (Braun and Cole 1984). Only the E. coli OmpA protein serves as a receptor for phage K3, whereas OmpA proteins from E. coli, S. dysenteriae, and S. typhimurium serve as receptors in E. coli for phage Ox-2 (Braun and Cole 1984). Differences in the aa sequence of regions "25" and "110" or in all four loop regions may be responsible for the observed receptor activity for phage K3 and Ox-2, respectively (Braun and Cole 1984). From some of the OmpA-specific phages, host-range mutants have been isolated which differ from their wild-type parent phages in their requirements for receptor structures. The genetic alterations of many of these mutants have been characterized. Therefore, these mutants will be discussed in the section on receptor binding proteins.

239 out K N

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rsx

The Tsx protein functions as a nucleoside-specific channel (Hantke 1976; Krieger-Brauer and Braun 1980). It serves as a receptor for the T-even-type but genetically different phages T6, H3 and Oxl and for colicin K (Hantke 1976; Manning and Reeves 1976). Tsx mutants which were solely impaired in their phage receptor function were obtained through selection for resistance against either of phages T6, H3, or Oxl. Screening was facilitated by placing lacZ immediately downstream of tsx and screening for T6r/lacZ +. This procedure excluded t s x - mutations with polar effects. All mutations obtained were located within the region of tsx encoding aa between positions 229 and 254 (Maier et al. 1990; Schneider and Bremer, unpublished) of the 272-residue Tsx protein (Bremer et al. 1990). The mutations comprise transversions (N249K, N254K, N254Y), deletions (aa 239-244), and duplications (aa 229-237). Most of these mutations were independently picked up several times (Schneider and Bremer, unpublished). They exhibit resistance against all Tsx-specific phages with the exception of T6h3.1. The latter phage was isolated from T6 as a host-range mutant able to infect cells producing TSXN254Y(Tsx carrying the N254Y mutation) (Maier et al. 1990). It also infects cells producing TSXN254K; however, cells producing other Tsx derivatives are resistant. T6h3.1, therefore, appears to be mutated in the gene encoding the receptor-binding protein. The finding that T6 r mutations are clustered in one region of Tsx is complemented by sequence analyses of tsx genes from other organisms, which show that this region is highly variable among the tsx genes from E. coli, S. typhimurium, K. pneurnoniae, and Enterobacter aerogenes (Nieweg and Bremer, unpublished). Receptor-binding proteins In this section I will focus on the analysis of receptorbinding proteins interacting with the receptor proteins

Fig. 1. Two-dimensional model of the N-terminal 175 aa of OmpA. Possible membrane-spanning /~sheets are boxed; positions of aa within the polypeptide chain are indicated only for the first and last aa of each/~-sheets; aa substitutions resulting in phage resistance are indicated; - , delection; 8 aa, insertion of eight aa.

described in the previous chapter. In particular the receptor-binding proteins of phage lambda, T5, and T-even phages will be discussed. More information about the phage appearing in this chapter can be found in the following reviews and books (Hendrix et al. 1983; Mathews at al. 1983; McCorquodale and Warner 1988) Lambda

Phage lambda is one of the best studied phages and its receptor protein LamB, one of the best studied outer membrane proteins. In contrast, the phage proteins involved in receptor recognition are only poorly characterized (Beumer et al. 1984). This is rather surprising, since the nucleotide sequence of the entire phage was published ten years ago (Sanger et al. 1982). The receptor-binding protein of phage lambda is most likely gpJ which is supposed to form the central tail fiber (Katsura 1983). Evidence for this stems from the following genetic and biochemical data: i) gpJ is the target of lambda-neutralizing antibodies (Buchwald and Siminovitch 1969); ii) host-range mutants of lambda map within the 3' end of gene J (Shaw et al. 1977); iii) LiCl-treated lambda ghosts are devoid of the tail fiber and proteins gpJ and gpH (Konopka and Taylor 1979); and iv) electron microscopic analyses of complexes between lambda and purified LamB protein show that binding to LamB occurs with the tip of the tail fiber (Roessner et al. 1983). Lambda forms two different types of complexes with either small LamB receptor particles or LamB protein incorporated into liposomes. In type I complexes, which most likely result from reversible interaction between lambda and LamB, binding appears to occur near the end of the tail fiber. In type II complexes, the distal end of the tail tube is directly attached to the receptor particles or the liposomes. This direct association is irreversible and appears to be a prerequisite for DNA ejection. An intermediate between type I and type II, an irreversible

240 type I complex, is formed at 4 ~ in 2 mM Mg 2+ using either E. coIi LamB in the presence of chloroform or Shigella Lamb in the absence of chloroform. Under these conditions no D N A ejection is observed. However, the intermediate complexes can be converted to mostly empty-headed type II complexes by a temperature shift (Roessner et al. 1983). The transition from type I to type II complexes is associated with increased protease sensitivity of gpJ, indicating a conformational change of gpJ (Roessner and Ihler 1984). It is not clear to date whether this conformational change represents the mechanism by which the tail tube is brought into contact with the cell surface or whether it is just a mechanism to move gpJ out of the way. T5 T5 and the closely related BF23 are clearly distinguished from other morphologically similar phages, e.g., lambda, by their two-step D N A injection mechanism (McCorquodale and Warner 1988). But they also appear to be unique among the phages described in this chapter with respect to the location of the receptor-binding proteins. Although both phages contain two different kinds of tail fibers (Fig. 2), neither of these fibers is involved in binding to the FhuA or BtuB receptor protein. The two kinds of fibers serve the following functions inT5. The L-spaped tail fibers, formed by tail protein pbl (Saigo 1978) and encoded by the Itf gene (Heller and Krauel

T 100nm

(

pb6

pbl (LTF) conicalpart >'~ ~ ' ~ l ~ , pb5 (Oad) S I I pb2 (STF) )'U Fig. 2. Schematicdrawing of phage T5. Only one of three L-shaped tail fibers (LTF) is shown. The receptor-binding protein pb5 is indicated at the transition from the conical part to the straight tail fiber (STF)

1986; are involved in the non-essential, reversible binding to polymannose O antigens (Heller and Braun 1979, 1982). The straight tail fiber, formed by tail protein pb2 (Heller and Schwarz 1985) and encoded by gene D18 (Zweig and Cummings 1973), is most likely involved in the penetration of T5 D N A through the E. coli cell envelope (Feucht et al. 1990). The receptor-binding protein pb5 (Heller 1984) is located at the transition from the conical part of T5 to the straight tail fiber (Heller and Schwarz 1985) (Fig. 2). pb5 of T5 is encoded by the oad gene (Krauel and Heller 1991). The term oad stems from a T5 mutant impaired in the interaction between pb5 and FhuA, resulting in an O antigen dependent phenotype (Heller and Bryniok 1984). Interestingly, the bacteriophage BF23 hrs gene, which also encodes the receptor-binding protein pb5, does not show any D N A homology to its T5 oad counterpart (Krauel and Heller 1991), although both gene products can functionally replace each other in phage assembly (Heller 1984; Krauel and Heller 1991). The peculiar location of pb5 in the tail implies that the straight tail fiber and not pb5 interacts first with the cell surface. However, no data exist which support the involvement of the straight tail fiber in receptor recognition (Heller and Schwarz 1985). This distinguishes the T5 tail fiber from that of lambda, although both appear morphologically similar, In lambda, type 1 complexesare formed between the tip of the tail fiber and LamB receptor particles (Roessner et al. 1983); such complexeshave never been describedfor T5. The complexesbetween T5 and FhuA receptor particles always resemble the type 2 complexes described for lambda (Heller and Schwarz 1985, Roessner et al. 1983). However, while gpJ of lambda becomes protease sensitive in type 2 complexes formed with liposome-incorporated Lamb (Roessner and Ihler 1984) pb2 of T5 becomes protease resistant in complexes formed with liposome-incorporated FhuA (Feucht et al. 1990). This indicates that, in contrast to gpJ, pb2 has become inserted into the liposomes when binding of pb5 to FhuA takes place. The receptor-binding domaine ofpb5 ofT5, consisting of 640 aa (Krauel and Heller 1991), is confined to ca. 200 aa within the N-terminal half. The C-terminal half and possibily 90 aa at the N terminus are most likely needed for the assembly of pb5 into the tail. Evidence for this comes from biochemical and genetic data (Mondigter and Heller, unpublished results): i) an overexpressed pb5 derivative, lacking the 152 C-terminal aa's, still binds to the FhuA receptor protein; ii) an internal deletion of aa residues 93 to 142 abolishes binding of pb5 to FhuA; iii) the oad mutation maps between aa residues 160 and 220; iv) the pb5 proteins of T5 and BF23 share only some relatively small regions of aa homology, mostly within their C-terminal halves. The latter finding clearly distinguishes T5 and BF23 from all the other phages described in this chapter. Usually, closely related phages differ significantly only in the portion of the receptorbinding protein responsible for receptor recognition. This is definitely true for the T-even and the lambdoid phages. The receptor-binding proteins of T5 and BF23, however, appear to be totally unrelated with respect to the coding D N A sequence, indicating that both genes are of different

241 genetic origin (Krauel and Heller 1991; Mondigler and Heller unpublished results). Binding of T5 to its FhuA receptor is very strong: centrifugation of T5-bacterium complexes, which are arrested at the stage when just 10% of the T5 genome has been transferred to the bacterium, causes the heads of the adsorbed phage particles to be broken away from their adsorbed tails but does not cause the entire phage particle to be released from the cell surface (Labedan et al. 1973). Based on the high activation energy measured for irreversible binding between T5 and isolated receptor particles Zarnitz and Weidel (1963) proposed the formatmn of covalent bonds between the phage and the receptor. However, recently it has been shown that covalent bonds are formed between three copies of tail protein pb4 rather than between pb5 and FhuA (Feucht et al. 1989).

T-even-type phages Each of the T-even-type phages binds to two different receptors on the bacterial cell surface in a sequential manner. The first, reversible step is mediated by the long tail fibers which bind to lipopolysaccharide or to outer membrane proteins. It is this step that determines the host-receptor specificity of the T-even phages (Beumer et al. 1984). However, for T4 it has been shown that viable phages lacking the long tail fibers can be isolated (Crowther 1980). In the second, irrreversible step the short tail fibers bind to lipopolysaccharide (Beumer et al. 1984). The lipopolysaccharide moiety they bind to may be either the outer core region including heptose (Riede 1987) or lipid A including keto-desoxy-octonate (Heller et al. 1983). In this section I will focus on phage proteins involved in the reversible step that determines host receptor specificity. Although the T-even phages represent a closely related group, heterogeneity between these phages has been demonstrated by immunological methods and D N A hybridization experiments (Schwarz et al. 1983,

i

r

/ ~

A.

\.-,..

gpl I (tailspike)

gp12 (shorttailfiber, storedposition)

Fig. 3. Schematicdrawing of a T-even-typephage belonging to the

T2 family. Only one of six long tail fibers is shown. Geneproducts forming the distal half of this fiber are indicated. The short tail fibers, involved in irreversible binding to LPS. are shown in the stored position; gp38 is absent in T-even-type phages of the T4 family

Riede et al. 1984). All T-even-type phages bind to their receptors with the tips of the long fibers (Beumer et al. 1984), but two different proteins are employed in this reaction. Phages of the T4 family (T4, TuIa, TuIb) bind to their receptors via the free ends of gene product 37 (gp37) (Montag et al. 1987) which, as dimers, form the distal parts of the long tail fibers (Beckendorf 1973; Beckendorf et al. 1973). Phages of the T2 family (T2, K3, Ox2, M1) bind to their receptors via gp38, which is present in one copy at the distal ends of the long-tail fibers (Fig. 3), thus acting as an adhesin (Riede et al. 1987b; Montag et al. 1987). The nucleotide sequences have been determined for the entire gene 37 o f T 4 (Oliver and Growther 1981), for the T-terminal ca. 900 nucleotides including the host-range determinants o f genes 37 o f phages T u l a and TuIb (Montag et al. 1990), and for genes 38 of phages T2, K3 (Riede et al. 1987a), Ox2, and M1 (Montag et al. 1987). In addition, the nucleotide sequences of genes 38 from host-range phage of K3 (Riede et al. 1987a) and Ox2 (Drexler et al. 1989, Drexler et al. 1991) have been determined. The aa sequences of the host-range-determining segment of gp37 of T4, TuIa, and TuIb, and of the lambda Stf protein, a component of the side-tail fibers (Montag et al. 1989). have been compared (Montag et al. 1990). The region responsible for receptor recognition appears to be confined to 70 to 100 residues of variable sequences located 43 to 44 residues upstream of the C terminus. The flanking regions show a high degree of homology. The variable sequences are flanked and interrupted seven or eight times by a His-x-His-y motif, with x and y predominantly being Ser or Thr. The locations of these repeats are conserved (Montag et al. 1990). A similar situation exists with gp38 of the T2 family. Alignment of the 260 aa polypeptides revealed two conserved regions, a 120 aa N-terminal and a 25 aa C-terminal region, divided by a hypervariable region of ca. 115 aa. This hypervariable region is flanked and interrupted by five highly conserved oligo-glycine residues (Montag et al. 1987; Riede et al. 1987a). The fact that mutational alterations affecting the host range of K3 and Ox2 are located within the hypervariable region indicates that this region is the receptor-recognizing region of gp38 (Riede et al. 1987a; Drexler et al. 1989; Drexler et al. 1991). The possibility of long-range effects of these mutations (unmasking of a cryptic binding site at a distant location on the polypeptide chain) can be almost excluded. In the Ox2 host-range phage, Ox2h 10, the H 170R mutation resulted m the phage's ability to brad to OmpC in addition to OmpA. The R170S alteranon in Ox2hl2hl.ll occurred at the same position, however, with this phage it resulted in the ability of the phage to bind to LPS (Drexler et al 1991). The G 121GG alteration, leading to host-range mutant Ox2hl 2, affected the first conserved oligo-gylcine stretch. The fact that this alteration was later lost during isolation of subsequent host-range phages may be taken as evidence for the indirect action of this alteration (Drexler et al. 1991). Among the Ox2 host-range phages isolated, two with different alterations, GI72T and the delectmn of A 198, showed the same phenotype. This may be taken as evidence that the four hypervariable regions between the oligo-glycine stretches interact with each other, thereby forming the receptor recognition area (Drexler et al. 1989; Drexler et al 1991).

242

One of the very fascinating aspects of receptor recognition by T-even phages is the enormous potential of mutational alterations resulting in different host ranges. This potential prompted Henning and his coworkers to discuss the phage-phage receptor system as a primitive immune system (Riede et al. 1987a; Drexler et al. 1991). By sequentially isolating host-range mutants, it was possible to successively change the receptor specificity of Ox2 from an outer membrane protein (OmpA) not only to other proteins (OmpC, OmpX) but also to lipopolysaccharide (LPS) (Drexler et al. 1991). However, this seemingly amazing result becomes less astonishing if one considers that phage T4 can bind to OmpC and LPS with equal efficiency and that the receptor-recognizing region is the same for both types of receptors (Montag et al. 1990). Mutations leading to different host ranges may have evolved by different mechanisms. Besides spontaneous events, recombination with DNA's from different sources appears to be involved. So far it has been shown that i) recombination of phage K3 D N A with cloned D N A from genes 23 (encoding the major head protein) and 37 (encoding the proximal half-fiber protein), respectively, can change the K3 host range (Riede 1986; ii) recombination between T4 D N A and D N A encoding the side tail fiber of phage lambda can functionally replace the receptor-recognizing part of the long tail fibers of T4, resulting in an altered T4 host-range (Montag et al. 1989); iii) chromosomal D N A of E. eoli K-12 hybridizes with D N A encoding parts of phage T2 tail fiber genes and can recombine into the D N A of other phages, e.g., K3, when present on multi-copy plasmids during infection (Riede et al. 1985b). A thorough analysis of nucleotide sequences of tail fiber genes further indicates that horizontal transfer of these genes occurs among unrelated bacteriophages (Haggard-Ljungquist et al. 1992).

The genetic region of T4-type phages comprising the 3'-end of gene 37 and gene 38 is reminiscent of the host-range "cassettes" found in phages P1 and Mu (Snyder and Wood 1989). Both these phages can alternate between two host-ranges by inversion of a D N A segment: the C region of P1 and the G region of Mu (for a review, see Koch et al. 1987). The invertible region in Mu includes the 3'-end of the gene encoding the C terminus of the tail fiber protein as well as a gene encoding an accessory protein involved in the assembly of the tail fiber. Comparison of the nucleotide sequence of T4 gene 37 around the boundary of T4/T2 homology revealed similarity to the recombination sites of Mu and P 1 G and C regions and, in addition, to the hin (H inversion) system of S. typhimurium (Snyder and Wood 1989). So far, there is no evidence that inversion events play a role in establishing host ranges of T-even phages. The significance of the findings of Snyder and Wood (1989), therefore, remains uncertain.

Receptor recognition Bacteriophages are usually very stable particles and are relatively resistant against high temperature, high salt concentrations, proteases, etc. However, phages must be considered metastable structures, with enough conforma-

tional energy stored to energize the first steps of infection. This is most impressively demonstrated with the T-even phages, where interaction of baseplate structures with appropriate receptor structures results in a conformational change of the major tail sheath protein. As a consequence the tail sheath contracts and generates force, which drives the tail tube through the outer membrane. That indeed force is generated is best seen by the fact, that upon contraction of T4 tail sheaths parts of the outer membrane are torn off bacteria with lowered OmpA content in the outer membrane (Zorzopoulos et al. 1979). Furthermore, it has been shown that the conformation of the major tail sheath protein of T4 is more stable in the contracted than in the extended state (Berget and King 1983). Irreversible phage adsorption may, thus, be considered as the transition from a metastable to a stable conformation. The energy required to trigger this conformational change is provided by the thermodynamically favorable binding of the receptorbinding protein to the target area of the receptor. This section will cover phages of the T-even-type, since molecular data have been published only for these phages. The conclusions, however, are likely to be applicable for other phages. What happens, if the target area of the receptor has been altered by mutation such that the cells carrying the receptor have become resistant to the phage? In this case, improper binding of the receptor-binding protein to the receptor prevents overcoming of the activation energy barrier and triggering does not occur. Selection for phages able to infect such cells yields, basically, two types of host-range mutants: i) phages with new host ranges, and ii) phages with extended host ranges. Phages of the first type differ in their host ranges from their immediate progenitors. They have altered receptorbinding proteins which can gain the activation energy needed for triggering by thermodynamically favorable binding to the mutated receptor proteins. Since only a very limited number of mutational events can cause this new host range, the frequency of such mutations is expected to be very low. An example of this type is K3hx (Riede et al. 1985a). It was isolated as a mutant of the OmpA-specific phage K3 that was able to infect strains carrying an OmpA derivative with D105-T106 deleted. K3hx was found with a frequency of 2 - 4 x 10 - l ~ among K3 wild-type phages. The mutation was shown to be a duplication ofK164-L165 ofgp38 of K3 (Riede et al. 1987a). Besides the OmpA deletion derivative, K3hx can use wild-type OmpA as receptor. However, in contrast to K3 which can use OmpA derivatives with alterations within loops 1 or 3, K3Hx fails to infect strains carrying these OmpA derivatives (Riede et al. 1987a). Alterations within loop 4 are tolerated by K3 as well as K3hx. This may be taken as evidence that loop 4 of OmpA is not involved in the formation of the receptor area for K3 (see chapter on OmpA).

For the receptor recognizing domain of gp38, a model has been presented which shows this region as consisting of four loops (Drexler et al. 1989; see section on T-even-type phages). Interaction of all four loops of gp38 of K3 with the target sequences on OmpA (loops 1, 2, 3) provides activation energy high enough for triggering.

243 For wild-typeK3, proper interactionwith loops 2 and 3 appears to be sufficientfor overcomingthe activation energy. For K3hx, which alreadyinteracts improperlywith loop 3 of wild-typeOmpA (the site of the D 105-T106 deletion), additional loss of interaction with loop 1 leaves an energybarrier too high for triggering. Based on the "allelespecificity"of the K3hx gp38, the conclusioncan be drawn that loop 2 of the receptor recognizing domain of gp38 interacts directlywith surface-exposedloop 3 of OmpA. It has been suggested that oligo-glycine stretches increase the flexibility of the receptor-recognizing areas and thus facilitate proper orientation and binding to the receptor (Riede et al. 1987a). However, such flexibility is hard to reconcile with the proposed metastable conformation of proteins involved in receptor recognition and triggering. Instead, the interpretation of the hypervariable regions separated by oligoglycine stretches as compact Omega-loops (Drexler et al. 1989) appears more appropriate. Phages with extended host ranges fall into two classes: i) phages with mutations outside the gene encoding the receptor-binding protein (in the T-even-type phages these mutations affect genes encoding components of the baseptate (Goldberg 1983; Drexler et al. 1989)); and ii) mutations affecting the gene encoding the receptorbinding protein. The first class, known as "trigger happy", may be considered as phages which require a lower activation energy for triggering the conformational change of the baseplate. Due to an increased lability of the baseplate, proper or coordinate binding of the long tail fibers to the receptor is no longer essential for triggering (Goldberg 1983). This explains the ability of some of these phages to infect cells with very low receptor densities: while wild-type phages require binding of at least three symmetrically arranged tail fibers, trigger happy phages already trigger after binding of one fiber (Goldberg 1983). The tall fiber-less mutants of T4 represent an extreme case of trigger happiness, where no binding to the receptor at all is needed for triggering (Crowther 1980). These mutants are extremely unstable and undergo baseplate transitionsin the absence of host cells. In general,trigger-happyphagesare characterizedby their increased sensitivity to elevated temperatures (Drexler et al. 1989), their extended rather than new host-range (Drexler et al. 1989, 1991), their ability to plate on cells with low receptor densities (Morona and Henning 1984, Drexler et al. 1989), and the relatively high frequency (10.6 to 10-3) of occurence (Crowther 1980; Drexler et al. 1989). The second class of phages with extended host range is less clearly understood. From the analyses of hostrange mutants of K3 and Ox2 that were able to recognize OmpA derivatives (Morona and Henning 1984; Riede et al. 1985a, 1987b), it appears that extended host range is caused by reduction of the requirement for proper interaction with the surface-exposed loops of OmpA. However, how can this be achieved by mutations within the receptor-binding domain of gp38? An explantation could be that the mutations relieve the structural constraints imposed on gp38 upon binding to altered OmpA carrying mutations within the loops forming the receptor recognition area. One such mutation in gp38 could restore binding to different OmpA derivatives, altered in the same loop or, more general, in the same target area.

However, this does not explain how the activation energy barrier is overcome to initiate triggering. It is very unlikely that the mutations in gp38 not only relieve the structural constraints during binding but also facilitate the conformational changing of the tail fibers, thereby lowering the activation energy needed for triggering. What has been found in fact is that all phages with extended host ranges have aquired additional mutations resulting in trigger happiness (Drexler et al. 1989). The molecular structures mediating the extension of the Ox2 host range from OmpA to LPS remain unknown. The LPS-specific Ox2 host-range mutants, carrying mutations in gp38, are all of the trigger-happy type, carrying additional mutations outside of gene 38 (Drexler et al. 1989, 1991). However, by complementation and recombination experiments the host specificity was clearly shown to be attributable to the mutations in gp38 (Drexler et al. 1991). Phage Ox2 obviously represents an ideal model system to study the evolution of different host specificities among T-even phages of the T2 type. All wild-type phages are very stable particles which survive harsh treatment, while the isolated Ox2 host-range mutants are rather labile particles (Drexler et al. 1989). It is tempting to speculate that during evolution, mutations resulting in triggerhappiness are transiently needed to allow the sequential accumulation of gp38 mutations, since the probability of these mutations to occur in one step is very low and, may thus be considered unlikely to occur outside the laboratory. It would be interesting to see whether stable, LPS-specific Ox2 derivatives can be obtained by selection for temperature resistance.

DNA injection Injection of phage DNA into bacterial cells provided the final proof for DNA being the genetic informationcarrying molecule (Hershey and Chase 1952). However, the molecular mechanism of DNA injection into the cell still remains an open question. A detailed discussion of this mechanism would certainly exceed the scope of this review. I will, however present the very recent findings concerning the functioning of phage proteins as channels for the penetration of phage DNA across the cell envelope. Bacteriophage infection is associated with permeability changes of the cytoplasmic membrane. This is generally understood as the insertion into the membrane of a pore protein carried by the phage. Two models of this event are currently discussed: i) depolarization of the cytoplasmic membrane eliminates the barrier raised by the membrane potential for the uptake of the polyanionic phage DNA (Keweloh and Bakker 1984); ii) the pore inserted into the cytoplasmic membrane acts as a diffusion channel for phage DNA (Letellier and Boulanger 1989). Recent findings with phage T5 are in favor of the second model, i) The purified protein forming the tail tip has been shown to form pores when reconstituted into black lipid membranes (Feucht et al. 1990). ii) From

244 the conductance data the diameter of the pore has been calculated to be 2 nm (assuming that the pore forms a cylinder of 7.5 nm length), which would be large enough to allow penetration of double-stranded phage D N A (Feucht et al. 1990). This diameter would also be consistent with data on potassium efflux from T5-infected cells, which indicate that the inserted pore is very large compared to other pore-forming compounds (Boulanger and Letellier 1988; Letellier and Boulanger 1989). iii) During infection the tail tip protein apparently becomes inserted into contact sites between inner and outer membrane (Guihard et al. 1992). I should mention that the concept of contact sites has been disputed throughout the last years (Kellenberger 1990), however, recent results presented by Bayer (1991) again support this concept, iv) The kinetics of potassium efflux from T5-infected cells match the kinetics of uptake of T5 D N A into the cells, which occurs in two distinct steps (Boulanger and Letellier 1992). v) Uptake of T5 D N A into host cells does not require any metabolic cellular energy (Filali Maltouf and Labedan 1983). The uptake ofT5 D N A into host cells, therefore, may be seen purely as a passive diffusion process through a proteinaceous channel. The channel, formed by five or six copies of the tail tip protein, either spans outer and inner membrane at contact sites or outer membrane, periplasmic space, and inner membrane at the site of infection. Except for the outer membrane receptor protein necessary for irreversible adsorption, no other host protein appears to be involved in the insertion of the phage channel into the host cell membranes. Can the uptake of T5 D N A be taken as a general model for the uptake of the DNA's of other doublestranded D N A phages? Although there appear to be important differences among the different phages, I think, yes. Two examples of phages which are apparently different from T5 with respect to D N A penetration shall be discussed. Phage lambda requires, in addition to the outer membrane receptor LamB, the inner membrane protein PtsM (Pel) for D N A penetration (Elliot and Arber 1978). A possible explanation could be that PtsM is an essential component of the DNA channel of lambda. However, since PtsM can be bypassed by mutations affecting either gpV or gpH (Katsura 1983), this possibility can be ruled out. The protein of phage lambda, forming the transmembrane channel for DNA uptake, has been suggested to be gpH* (Roessner and Ihler 1984). However, in contrast to the pore-forming tail tip protein of T5, gpH* apparently does not form the tip of the phage lambda tail, but may rather serve as a tape measure protein located inside the tail tube (Katsura and Hendrix 1984). Whatever the location of gpH* within the lambda tail may be, comparison of its deduced amino acid sequence (Sanger et al. 1982) with that of the T5 tail tip protein (Feucht, Demleitner and Heller, unpublished results) suggests some similarity between both proteins, indicating that they may serve similar functions. Formation and opening of the protein channel of phage T4 in the cytoplasmic membrane of the host cell requires an energized membrane, which has been inter-

preted in terms of voltage gating of the T4 channel (Letellier and Boulanger 1989). This certainly appears to distiguish the T4 channel from that of T5. However, in a model originally presented by Furukawa et al. (1983) and recently reinforced by Tarahovsky et al. (1991), the state of energization of the cytoplasmic membrane regulates its distance from the outer membrane: only in the energized state is the distance between outer and inner membrane small enough that it can be spanned by the tail tube. According to this model the T4 channel, once it has been formed, would be independent of the energy state of the inner membrane. Finally, I like to mention that the mechanisms discussed in this chapter are only applicable to the uptake of double-stranded phage DNA. For the uptake into the cell of the single-stranded D N A of filamentous phages a complex cellular machinery, involving tol gene products, is responsible (Sun and Webster 1986, 1987). However, penetration of the D N A through the outer membrane may be facilitated by g3p, the receptorrecognition protein which has been shown to form pores when reconstituted into artificial membranes (GlaserWuttke et al. 1989). Conclusions

Accumulation of molecular data on receptor recognition by phages over the last years has provided some insight into the underlying molecular mechanisms. Some common motifs emerge. The receptor-recognizing areas of phage proteins seem to be confined to single regions on the polypeptide chains. The repeat structure of these regions, as found in T-even-type phages, seems t o be specific for this group since it is not found in other phages such as lambda, T5, and BF23. The maximal number of surface exposed loops of receptor proteins recognized by the phages appears to be limited to four loops. The knowledge of X-ray structures and multiple alignment calculations of related receptor proteins will soon provide exact topological models of these proteins. These models will make possible the positioning of the receptor-binding proteins with respect to the surface-exposed loops involved in phage reception. The knowledge of the exact nature of mutations with new host-ranges will be of great value for such positioning. The TonB-dependent receptor proteins are a group of transport proteins which appear able to undergo considerable conformational changes not only during transport but also during phage adsorption. The isolation of receptor missense mutations impaired solely in phage reception should be very rewarding for TonB-dependent phages, since this could possibly lead to the identification of transiently surface-exposed loops of the receptor proteins. As for D N A uptake, the biochemical and biophysical characterization of pore-forming phage proteins should help elucidate the molecular mechanism of membrane penetration. Studies of this mechanism will be very much facilitated by the accessibilities of the corresponding genes to modern molecular biology techniques.

245 Acknowledgements. I thank E. Bremer, T. Ferenci, and L. Letellier for reporting unpublished results and E. Bremer and V. Koogle for their comments on the manuscript. I acknowledge the support by the Deutsche Forschungsgemeinschaft (He 1375/3-3).

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