the spp1 connection

ELSEVIER FEMS Microbiology Reviews 17 (I995) 47-56

MICROBIOLOGY REVIEWS

The SPP1 connection

Paulo Tavares a,,, Anja Dr/Sge a, Rudi Lurz a, Inge Graeber h, Elena Orlova c, Prakash Dube e, Matin van Heel c

a Max-Planck-lnstitutfiir Molekulare Genetik (Abt. Trautner), lhnestrasse 73, D-14195 Berlin, Germany b Bundesgesundheitsamt lnstitutfiJr Veteriniirmedizin, Diedersdorfer Weg 1, D-12277 Berlin, Germany

¢ Fritz Haber lnstitut tier Max-Planck-Gesellscha#, Faradayweg 4-6, D-14195 Berlin, Gerraany

Abstract

The connector of the virulent Bacillus subtilis bacteriophage SPP1 (Styloviridae) is a structure localized at the phage head vertex which attaches the tail. It is formed by oligomerization of SPP1 gene product 6 (gp6; portal protein). The purified protein is found in solution essentially as a homo-tredecamer. Its assembly pattern resembles the turbine-like organization found for other portal proteins and has a defined handedness (Dube et al. (1993) EMBO J. 12, 1303-1309). A preliminary reconstruction of the structure shows that gp6 is composed of a lower ring connected by a narrow region to the upper area consisting of 13 lobes radiating from an inner ring. The assembly is organized around a central channel which spans its full height. A functional characterization of gp6 mutants showed that substitutions of defined amino acids by more basic residues lead to packaging of reduced amounts of DNA into the phage head (Tavares et al. (1992) J. Mol. Biol. 225, 81-92). Since SPP1 encapsidates its DNA by a headful mechanism, these mutations (s i z ) affect most probably a function on the headful sensor-signal transduetion-headful cut system. Combination of siz alleles has severe effects in packaging. The resulting gp6 versions lead to the encapsidation of shorter DNA molecules at a lower efficiency than single siz mutants. Gene 6 is expressed late during SPP1 infection. Interestingly, the mass of portal protein inside the ceil then increases continuously until lysis, reaching a level several fold higher than the amount required to accomplish its role as a structural component of the virion.

Keywords: Bacillus subtilis; Bacteriophage SPP1; DNA packaging; Portal protein; Connector; Chromosome sizing

1. Introduction

A com mon feature found among tailed icosahedral bacter iophages which encapsidate double- stranded D N A (dsDNA) is the presence o f a small knob structure located at the unique vertex o f the viral capsid where the tail attaches (connector [3];

* Corresponding author. Tel.: +49 (30) 8413 1265; Fax: +49 (30) 8413 1385

Fig. 1). T h e main component of this s t ruc tu re is a homo-ol igomer named portal protein [4]. Although additional proteins might also be part o f the knob observed in phage particles (Fig. 1), the functional concept o f connector is usually restricted to the portal ol igomer. This is due both to the relatively good knowledge of portal protein architecture and to the essential roles it was shown to play in different phases during viral assembly [5] while the structure and function of the other polypeptides associated to the portal vertex remain less characterized.

The portal vertex structure, which differs f rom the

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48 P. Tavares et aL / FEMS Microbiology Reviews 17 (1995) 4 7 - 5 6

other 11 vertices of the icosahedral pro-head, is essential for the mechanism of DNA packaging. During this process a nucleoprotein complex ( terminase-DNA) is believed to bind to the portal area leading to DNA translocation into the pre-formed head shell, a movement occurring most probably through the connector pore ([4]; Fig. 2). A mechanistic hypothesis proposes that the connector itself, associated to the terminase, could act as an ATP- driven molecular motor pumping the D N A against a free energy gradient [1,3]. The end of encapsidation usually involves cleavage of the packaged DNA molecule, determining chromosome size (section 3), binding of additional proteins, and attachment of a tail to the portal vertex [6].

The connector topology in the head shell (Fig. 1) fits well to the roles it plays in viral maturation such as the recogni t ion/docking of other structures ( te rminase-DNA complex, tail) and probably also mediating information exchange between these other structures and the viral capsid. Moreover, the molecular architecture o f the portal assembly, with its central channel ([1,5]; Fig. 2), suggests an important role supporting DNA movements into and out of the phage head.

Characterization of the portal protein from SPP1 (gene product 6, gp6; [2]), a Bacillus subtilis bacteriophage which packages its DNA by a headful mechanism, yielded two main new findings. Struc- tural studies by electron microscopy and image processing showed that purified gp6 is an oligomer with 13-fold cyclical symmetry [1]. Functional and genetic analyses demonstrated that it plays a central role in viral chromosome size determination [2]. In this paper we review these and other recent advances in SPP1 portal protein research. Studies on analogous proteins will only be referred to when of direct relevance to the SPP1 system. For a detailed overview of the 'state of the art' on portal protein research and its connection to general phage physiology and assembly, we refer the reader to several comprehensive reviews [4-8].

2. Structure of the SPP1 portal protein

The SPP1 portal protein gene was cloned into a plasmid and the recombinant construct was used to

transform Escherichia co i l Gp6 was purified from this overproducing strain. It is an acidic protein found in solution essentially as a large homo- oligomer ( M r ~ 700-800 kDa; [9]). Electron microscopy studies in combination with image processing demonstrated that gp6 molecular architecture resembles the overall structure of other portal proteins. Processed top views of gp6 show the presence of thirteen handed lobes radiating from a central ' tubular ' channel [1]. This turbine-like assembly has a clear radial 13-fold symmetry in contrast to what was previously found for other bacteriophage portal proteins which were described as homo-dodecamers (reviewed in [5]; see also below).

More detailed structural information became recently available from a three-dimensional reconstruction of the portal protein ( ~ 20 A resolution) based on processing of different views of the oligomer ([10]; Fig. 2). The reconstruction shows that gp6 is composed of a lower doughnut (98 ~, diameter) connected by a narrow area to the upper wide region (175 ,~ diameter) which includes one ring that delim-

Fig. I. Cryo-electron micrograph of frozen-hydrated SPP1 particles. Some viruses lost their DNA, probably after adsorption to bacterial debris, allowing easy visualization of the connector (arrows). The bar represents 100 nm. Samples were frozen in liquid ethane and ~hotographed at --163°C with an electron dosage of 8,5 e - / A z in a Philips CM100 electron microscope equipped with a cryo holder from Oxford Instruments.

P. Tavares et al. / F E M S Microbiology Reviews 17 f l995) 4 7 - 5 6 49

its the central channel and 13 independent p ro t rud ing lobes. A cont inuous channel spans the full height (105 ,~) o f gp6. The ol igomers overall shape r e sem- bles the mode l s o f other connectors (¢k29 [ 1 1 - 1 3 ] and T3 [14,15]). Al though the reconstruct ion o f the SPP1 portal protein is still pre l iminary , re levant dif- f e rences with respect to other publ ished s t ructures m a y a l ready be poin ted out. In addition to the s y m - me t ry d i f ference and sl ightly larger d imens ions o f the SPP1 portal o l igomer , gp6 has a na r row region separa t ing the two coaxial r ings (side v i ew) wh ich is absent in ~b29 and "I"3 connectors . T h e uppe r and

lower sur faces o f the SPP1 structure are also di f ferent f r o m the flat regions shown in both the ~b29 [11,12] and T3 [14,15] reconstruct ions .

The f inding o f 13-fold s y m m e t r y in the SPP1 and ~b29 por ta l proteins [1], as opposed to the "class ical ' 12-fold organiza t ion o f connectors , genera ted s o m e cont roversy . It was p roposed that the me thods initially used to s tudy portal a s sembl i e s were b iased towards even-fo ld or specif ical ly towards 12-fold symmet r i e s , thus p reven t ing the ident if icat ion o f 13- fold s y m m e t r i c c o m p o n e n t s [1]. A d i f ferent poin t o f v i ew is that the m a s s i v e ove rp roduc t ion o f por ta l

Fig. 2. Three-dimensional structure of the SPP1 portal protein based on cryo-eleclron microscopy of negatively stained gp6. Upper part: A stereo pair of the portal protein seen from above (tilted towards the viewer by 10°). The 13-fold symmetry of the structure is evident. Lower part: A stereo pair of the gp6 side view. Experimental procedure: A large number of gp6 molecular images (~- 6000)was selected from a set of digitized electron micrographs and processed using the IMAGIC-5 image processing system [32]. Multivariate Statistical Analysis (MSA [33]), data compression and automatic classification [34] were applied to find the characteristic views of the portal protein [1]. The relative orientations of these average images with respect to each other, in three-dimensional space, were found using the angular reconstitution technique [35,36]. Once these orientations are known, the portal protein can be reconstructed using the 'exact-filter' back-projection technique [37]. A detailed description of this reconstruction and of the full procedures will be presented elsewhere.

50 P. Tavares et aL / FEMS Microbiology Reviews 17 (1995) 47-56

polypeptides in absence of other phage proteins could lead to oligomerization states different from the ones observed during infection and thus account for tl~e production of 13-fold symmetric oligomers which were invariably purified from overproducing strains ([5] and references therein). We find it premature at

this point to decide which is the prevalent symmetry of the portal protein during morphogenesis. A clearer picture will undoubtedly emerge when new studies on the connectors state of oligomerization at different steps of the virion assembly pathway will become available.

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Fig. 3. DNA encapsidation and genome organization o f bacteriophage SPP1. (A) DNA packaging. The SPP1 packaging apparatus recognizes and eneapsidates different substrates. The two types mentioned in the text are represented. Upper part: chimeric coneatemer of SPP1 DNA, including pac, and a series of tandemly repeated plasmid units organized in a head-to-tail fashion. This substrate is generated and packaged at high frequency when a plasmid resident in the infected cell carries homology (white boxes) to the infective SPPI DNA (facilitated transduction [24,25]). Plasmid DNA regions non-homologous to SPP1 are shadowed and the thick vertical bar identifies pac. Lower part: SPP1 concatemeric DNA. Mature chromosomes derived from one packaging series initiated at the right most pac sequence of the concatemer are shown below. The DNA molecules generated are partially circularly permuted and terminally redundant. Black boxes represent the major dispensable region within SPP1 genomc [29]. (B) Mature chromosome features. Upper part: Sizes of packaged DNA molecules determined by the s/z allele present during eucapsidation [2]. Lower part: EcoRl restriction map of SPP1 chromosome [38]. For clarity, not all fragment numbers are depicted. (C) Organization of the region coding SPP1 gone 6 and neighbouring area. Physical and genetic maps are based on sequence data from Chai et all. [21] and Tavarcs et al. [2]. Genes and open reading frames (off) are depicted by arrows. Restriction sites: A, Asp718; B, Boll; Bs, BsaHl; E, EcoRI; H, HindIII; Hp, HpaI; S, SalI. (H) indicates the position of an additional HindIII site generated by the mutation s/zA.

P. Tauares et al. / FEMS Microbiology Reviews 17 (1995) 47-56 51

3. Function of the portal protein in SPPI chromosome sizing

SPP1 packages its DNA by a headful mechanism ([16-18]; Fig. 3A). The concatemeric substrate DNA, generated by replication of the SPP1 chromosome, is initially recognized and cleaved within a specific sequence (pac ) by the terminase (products of gene 1 and 2) [19-21]. Af t e r translocation of a defined amount of DNA into an empty pro-capsid, a second nucleolytic cut occurs defining the mature chromosome size (Fig. 3A, B). The headful cleavage is sequence-independent and shows some imprecision, varying by more than 200 bp [22]. Packaged DNA molecules are (usually)terminally redundant. This property ensures that all essential phage genes are packaged in the virion and that the chromosome can circularize following infection. The encapsidation process of each individual substrate concatemer is unidirectional and processive, yielding an average of 3 -5 packaging events following the initial cleavage at pac [18,21]. As a consequence of this strategy of DNA maturation, a heterogeneous population of partially circularly permuted and terminally redundant chromosomes is generated (Fig. 3A,B).

The mode of packaging described requires that a headfilling sensing mechanism is operative in order to determine the occurrence of a nucleolytic cleavage after a defined amount of DNA is packaged inside the phage head. Several missense mutations (siz) in the SPP1 portal protein were shown to cause undersizing of packaged DNA with a concomitant reduction in headfilling of the mutant phage eapsids re- vealing, therefore, an alteration in such measuring function [2]. Three independent siz loci were identified within gene 6 (Fig. 3C) demonstrating tile main role of its product in this mechanism. Significantly, Casjens and his collaborators [23] characterized two different mutations in bacteriophage P22 portal protein coding gene (gene 1) which lead to ovemized chromosomes by affecting also the system that regulates headful cleavage.

Conservation of siz mutations within the phage genome is only compatible with viability when, in spite of the reduction in chromosome size, the full ensemble of essential viral genes and some terminal redundancy (necessary for chromosome cireulariza- tion after host infection) are still present in the

packaged DNA. This reqluirement can be accom- plished both in case of sh0~tt undersizing phenotypes or combination of the siz rautation with deletions in dispensable areas of the genome ([2]; Fig. 3B). A more detailed characterization of the original and newly engineered siz alleles could be obtained by using an in vivo test for a~saying the effect of any version of gene 6 in SPP1 maturation independently of its effect on phage plqysiology (Fig. 4A). It is based on the observation that, during an infection under non-permissive conditions, gene 6 suppressor sensitive mutants (sus) can be complemented in trans by a gene 6 allele l~rovided from a plasmid present in the host cell. Since the phage-encoded gp6 is a truncated, non-functio~aal protein, progeny phenotypes can be directly correlated to the plasmid gene 6 version. This strategy allows comparison of different siz alleles in an i~ogenic background (iden- tical infective phage and host) ([2]; Fig. 4) including those that produce a lethal phenotype and cannot, therefore, be established ha the phage genome.

Characterization of the progeny from a eomple- mentation assay includes biochemical, biophysical and biological studies. In t ie latter case, both infec- tivity and facilitated plasnnid transduction capacity ([24,25]; Fig. 3A and legend) can be scored providing distinct information. While a reduction in viable virus titer might reveal (i) encapsidation of an in- complete genome due to ur~dersizing (Fig. 313) or (ii) lower packaging efficiency, only the second situation would determine a decreage hn plasmid transducing particles since their mature DNA would always be a concatemer of several plast'aid units (Fig. 3A). Such analysis combined with electron microscopy and pulse field gel electrophoxesis of encapsidated DNA molecules showed that nl,ast single s/z mutations determine a short chrom~salne size with no major effects in the efficiency of packaging ([2]; Fig. 413).

The three siz mutations originally identified lead to single amino acid substitutions in distinct regions of gp6 (Fig. 3C). lnterestimgly, all of them involve changes to lysine residues (sizS: E~I ~ K; sizA: N365 ~ K and sizX: E424 ~ K) which cause, invariably, an alteration in charge. This correlation between chromosome size arid charge of gp6 specific amino acids was farther corroborated using in vitro mutagenesis. The phenotypes associated to one engineered mutant (sizX50) are shown in Fig. 4B. This

52 P. Tat~ares et aL / FEMS Microbiology Reuiews 17 (1995) 47-56

SPP1 sus 115

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P. Tavares et al. / FEMS Microbiology Reviews I7 (1995) 47-56 53

gp6 (l-tg) t (rain)

I 0.01 0.05 0.1 0.15 0 ,2 0.5 1.0 II 0 5 10 12 15 20 25 30 I

Fig. 5, Gp6 production during SPP1 infection of B. subtilis monitored by Western blotting. Left: Increasing amounts of purified portal protein. Right: Kinetics of gp6 production in infected cells (the time after infection is shown above). Gp6 has a mobility in SDS-PAGE corresponding to an apparent M r of 68 kDa, a higher value than 57.3-kDa which was predicted from gene 6 nucleotide sequence [2,9]. Experimental procedure: B. aubtilis strain YB886 [40] was infected with SPP1 wild.type (to; input multiplicity = 10) following our standard method [2] and at desired times 1 mt aliquots were mixed 1:1 (v/v) with ice-cold TBT buffer containing 10 mM sodium aside and 20% glycerol [20]. The cell suspensions were kept on ice until the last aliquot was taken and then immediately pelleted, resuspended in 40/.d TY medium [41] supplemented with 1 mg/ml lysozyme and lysed by "freeze and thaw'. Crude extracts samples (15 ILl) were separated by SDS-PAGE (15%) and gp6 was detected using anti-gp6 purified l eg (rabbit polyclonal) followed by anti-rabbit leG-alkaline phosphatase (Sigma) as secondary antibody and visualization with NBT-BCIP in diethanolamine buffer [28]. Blots were scanned with an Epson GT-8000 Scanner and digitized data was quantified using the program ImageOuant TM (Molecular Dynamics). Gp6 was purified as described [1,9].

gp6 vers ion carr ies an in termedia te a m i n o ac id sub- st i tution in te rms o f charge (E424 ---> Q) relat ive to the or iginal s i z X mutat ion . Signif icant ly , it leads to an in te rmedia te d imens ion o f the SPP1 c h r o m o s o m e w h e n c o m p a r e d to the s izes de te rmined b y wi ld - type and s i z X alleles (Fig. 4B). Severa l mu ta t ions wi th in the s i zS reg ion demons t r a t ed also a s imilar relat ion- ship b e t w e e n the charge o f res idues in pos i t ions 2 5 0 - 2 5 2 o f the portal prote in and size o f p a c k a g e d D N A (our unpub l i shed results). A m e c ha n i s t i c inter-

pre ta t ion o f this data will require, however , a m o r e deta i led charac ter iza t ion o f the different mutan t p ro - teins.

Mult iple mutants , obtained by combina t ion o f different s i z alleles, were observed to genera te p h a g e part icles con ta in ing signif icant ly shorter c h r o m o - somes than the wi ld - type vir ion (Fig. 4B). The level o f under s i z ing s e e m s to be dependent on the indiv id- ual unders i z ings associa ted to each allele present in the mult iple mutan t . Sco r ing for p lasmid t r ansduc ing

Fig. 4. Characterization of siz alleles. (A) Complementation assay. Experimental procedure: The version of gene 6 to be tested was cloned (segment of SPP1 DNA depicted in Fig. 3C) in plasmid pHP13 [39] and transformed into the non-permissive YB886 host [40]. This strain was then infected with SPP1 susl l5 (most amino terminus gene 6 mutant) and 70 rain later the lysate was cleared by centrifugation and treated with nucleuses as previously described [2]. Progeny particles (generated essentially from one single infection cycle) were then characterized. (B) Chromosome dimensions and biological properties associated to different s/z alleles. Equimolar amounts of packaged DNA molecules were resolved by pulsed-field gel electrophoresis (PFGE). Absolute size values based on electron microscopy measure- ments are also shown (left side). In several tracks a band co-migrating with the SPP1 wild-type chromosome can be seeu in addition to the main band (most evident in case of sizSsizAsizX). We attribute its appearance to the presence of revettants in the SPP1 sus115 parental lysate. The reproducible variability in chromosome size associated to sizSsizAsizX suggests a reduced accuracy in headful cleavage. However, we cannot exclude that recombination between the two gene 6 versions present in the infected eell(sus115 and sizSsizAsizX) might generate new mutant variants (e.g. sizSsizA; sizAsizX) at low frequency and their associated phenotypes, usually not detected, become evident in case of sizSsizAsizX due to the poor packaging efficiency determined by this gene 6 version (see rex0. In such case a population of packaged DNA molecules with variable sizes would also be expected. Biological activity was assayed by scoring the number of viable phages and plasmid-transducing particles. Results are presented as a comparison based on the titers determined for wild-type gene 6. WT, wild-type allele; S, sizS; X50, siz.gSO; X, s/zX; A, s/zA; X50S, sizXSOsizS; XS, sizXsizS; AS, sizAsizS; AX, sizAsiz~; SAX, sizSsizAsizX. Experimental procedure: The progeny lysate was treated with 50 mM EDTA at 55°C for 30 rain and extracted with phenol and chloroform. Free DNA molecules were dialysed and separated by PFGE in a 1.2% agarose gel (Seakem, FMC) using a GelNavigator TM apparatus (Pharmacia) with hexagonal electrodes and a 3-phases program (6 h each; pulses of 0.8, 1 and 1.2 s). Infection of a permissive strain and plasmid transduction were as previously described [2].

54 P. Tavares et a t / F E M S Microbiology Reviews 17 (1995) 47-56

particles (Fig. 413) and preliminary hybridization ex- periments (our unpublished results) showed that some of these multiple mutants lead also to a lower packaging efficiency.

The fact that mutations affecting chromosome size can influence packaging efficiency when combined together reveals an interdependent action of the corresponding portal protein domains during the maturation process. It also suggests a close mechanistic relationship between some step(s) of DNA eneapsidation (substrate recognition, translocation, efficiency of headful cleavage [8]) and chromosome size determination. This view is supported by the possibility that sizSsizAsizX might also cause a low accuracy of headful cleavage (see Fig. 4B and legend). Nevertheless, it should be kept in mind that the data presently available do not exclude that accumu- lation of siz mutations might also affect other steps in phage head assembly than DNA encapsidation. If this was the case, any effect leading to a reduction in formation of stable filled capsids would also con- tribute to the low packaging efficiency phenotype.

4. The SPP1 portal protein cistron

The portal protein coding gene (gene 6) was initially mapped within the SPPI genome following an attempt to identify a mutation responsible for the packaging of reduced amounts of DNA into the viral capsid (sizA [2]; Fig. 3C; previous section). Cloning and sequencing studies showed that it is localized downstream from the terminase cistrons (genes 1 and 2) and three open reading frames with unknown function [2,21], preceding an area of the chromosome where several structural protein genes were mapped [26]. This overall genomic organization is analogous to the one found in bacteriophages A and P22 and was suggested to be evolutionarily advanta- geous since it clusters genes whose products inten- sively interact [6,21,27].

As in other phage systems, the portal protein and terminase genes are co-transcribed from a late promoter with the levels of expression of each cistron being defined post-transeriptionally. Chai et al. [21] characterized the promoter, located immediately up- stream from gene 1, and could identify several RNA transcripts from this region observable 8 min after

SPP1 infection. The largest mRNA species (5.0 kb) was inferred to be a transcript of the region encompassing genes 1-6 [21]. The transcription start time of the terminase-portal protein operon correlates well with the expression of gene 6 whose product can be detected between 10 and 12 min after infection ([28]; Fig. 5). The amount of portal protein then increases continuously with time until cell lysis (approx. 30 rain). This pattern differs from the bell-shaped fate of mRNA level, which, when quantified by hybridization with a terminase-specifie probe, starts decreasing around 15 rain after infection [29]. Fur- ther studies will be required to distinguish whether this reduction is limited to the terminase region of the polycistronic mRNA or is also affecting the gene 6 area, with the increase of portal protein mass being assured by a highly efficient translation of the re- maining messenger.

Quantitative analysis showed that an average of approximately 130000 copies of portal monomers are present per infected cell (30 min). This value represents a large excess relatively to the theoretical amount of gp6 required to accomplish its structural role in the virion if we take into consideration the SPP1 burst size (260-560 infectious particles/cell depending on the experimental conditions [30]) and assume that the portal protein is also a 13-met in the phage particle [1]. The meaning of such high production of gp6 in infected cells is not yet understood.

5. Summary and discussion

The importance of portal proteins in tailed icosahedral bacteriophage assembly is well documented. Detailed analyses have been, however, limited by the absence of high resolution structures and by the difficulties in assessing the molecular mechanisms underlying the portal protein multifunctionality. This situation is presently being overcome by a revitalized interest in connector architecture and detailed characterization of different portal protein domains using site directed mutagenesis in combination with bio- chemicM and functional assays (reviewed in [5]). These techniques are also being applied to study the bacteriophage SPP1 system.

The SPP1 connectors overall organization (Fig. 2) supports the concept that portal proteins share a

P. Tavares et al. / FEMS Microbiology Reviews 17 (1995) 47-56 55

conserved turbine-like assembly motif, probably uni- versal among tailed icosahedrai bacteriophages which encapsidate dsDNA. It therefore seems reasonable to speculate that such an architecture is required to accomplish an essential function common to all these viruses, namely DNA translocation. The 'driving force' behind this process was suggested to be the symmetry mismatch between the connector and its environment (5-fold symmetric vertex) [3]. More recently, the intricate symmetry interactions between the portal protein and the DNA double helix were proposed to play a central role in the mechanism [1]. Direct correlations between the SPP1 portal structure and functional aspects are, however, not yet possible due to a lack of information both on gp6 domain topology within the portal oligomer and on conformational changes that occur in the connector during morphogenesis, as reported for the ~29 and T3 systems [12,14,15,31].

A more detailed picture on the role of specific portal protein amino-acids in chromosome size determination during SPP1 headful packaging is presently emerging. The charge of defined residues seems to be an important element in this process. Addition- ally, the combination of several mutations affecting size also reduces packaging efficiency, suggesting a mechanistic relationship between headful cleavage, which determines chromosome dimension, and other steps in encapsidation. Nevertheless, in absence of biochemical and structural data, the interpretation of these observations is still very limited. In a more general sense, some fundamental questions concern- ing the overall packaging mechanism remain poorly understood both in SPP1 and in other phages (see also [4,5,8]): how does the terminase-DNA complex recognize and bind the portal vertex? Which phage components are involved and how do they act in DNA translocation? Is the headful cut triggered by the impossibility of the encapsidation machinery to translocate more DNA into the phage head or are both processes mechanistically uncoupled (see also [23])? How can a single missense mutation in the portal protein affect headful cleavage? What mechanisms ensure the correct order of events avoiding, for example, tail attachment before DNA packaging? Thus, in spite of the significant recent progress in elucidating structural and functional aspects of the SPP1 portal protein, the detailed understanding of

the mechanisms of action of this assembly will require further concerted efforts of interdisciplinary research.

Acknowledgements

We are grateful to Prof. T.A. Trautner for criti- cally reading the manuscript. Valuable discussions and scientific support from Prof. T.A. Trautner, and Dr. J. Alonso and his group during the course of most of the work described are acknowledged. We thank Prof. E. Zeitler for his continuous support to the electron microscopy and image processing pro- ject. F. Zemlin and E. Beckmann are acknowledged for excellent negative staining cryo-electron microscopy. We are also grateful to Dr. M. Schatz and R. Schmidt for software and computing support. Furthermore, we thank Drs. J.M. Valpuesta and J.L. Carrascosa for kindly providing us with a manuscript prior to publication. P.D. and E.O. were partially supported by a DFG "Schwerpunkts Programm" grant ( # H E 21621-1) and a MPG post-doctoral Stipendium, respectively. P.T. was supported by an EEC fellowship (Contract ERBBIOTCL923104).

References

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[2] Tavares, P., Santos, M.A., Lurz, R., Morelli, G., Leneastre, H.L. and Trautner, T.A. (1992) Identification of a gene in Bacillus subtilis bacteriophage SPP1 determining the amount of packaged DNA. J. Mol. Biol. 225, 81-92.

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the spp1 connection

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