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Journal of Bacteriology, August 2005, p. 5397-5405, Vol. 187, No. 15
0021-9193/05/$08.00+0     doi:10.1128/JB.187.15.5397-5405.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Holin Protein of Bacteriophage PRD1 Forms a Pore for Small-Molecule and Endolysin Translocation

Gabija iedait,^1,2 Rimantas Daugelaviius,^1,2 Jaana K. H. Bamford,¹ and Dennis H. Bamford^1*

Department of Biological and Environmental Sciences and Institute of Biotechnology, Biocenter 2, P.O. Box 56 (Viikinkaari 5), 00014 University of Helsinki, Finland,¹ Department of Biochemistry and Biophysics, Vilnius University, iurlionio 21, 03101 Vilnius, Lithuania²

Received 6 December 2004/ Accepted 27 April 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
PRD1 is a bacteriophage with an icosahedral outer protein layer surrounding the viral membrane, which encloses the linear double-stranded DNA genome. PRD1 infects gram-negative cells harboring a conjugative IncP plasmid. Here we studied the lytic functions of PRD1. Using infected cells and plasmid-borne lysis genes, we demonstrated that a two-component lysis system (holin-endolysin) operates to release progeny phage particles from the host cell. Monitoring of ion fluxes and the ATP content of the infected cells allowed us to build a model of the sequence of lysis-related physiological changes. A decrease in the intracellular level of ATP is the earliest indicator of cell lysis, followed by the leakage of K⁺ from the cytosol approximately 20 min prior to the decrease in culture turbidity. However, the K⁺ efflux does not immediately lead to the depolarization of the cytoplasmic membrane or leakage of the intracellular ATP. These effects are observed only 5 to 10 min prior to cell lysis. Similar results were obtained using cells expressing the holin and endolysin genes from plasmids.

Top Abstract Introduction Materials and Methods Results Discussion References

 
For most bacteriophages, host cell lysis requires, at a minimum, two proteins: an endolysin and a holin. Endolysins are small enzymes that degrade cell wall peptidoglycan. These enzymes fall into four groups depending on their activity, which is directed against the three different covalent linkages that maintain the integrity of the cell wall: (i) glycosylase and (ii) transglycosylase activities targeting the glycosidic linkages, and (iii) amidase and (iv) endopeptidase activities targeting the oligopeptide cross-linkages (39). Most endolysins characterized to date have no signal sequence and therefore accumulate in the cytosol during infection. Holins are small hydrophobic integral membrane proteins that permeabilize the cytoplasmic membrane (CM) and allow the endolysins to attack the peptidoglycan (20, 38, 39). In addition, holins may work as activators of the endolysins (39). Holins are grouped into two classes based on their primary structure. Class I holins, such as bacteriophage S protein, generally have more than 95 residues and form three transmembrane helices. Class II holins are smaller (65 to 95 residues) and form two transmembrane helices (18, 39).

Holin-dependent lysis systems are highly regulated. The precise temporal regulation likely is dependent on the energy state of the CM, because adding a metabolic poison (e.g., cyanide or dinitrophenol) sufficiently late in the infection cycle instantly triggers cell lysis (14, 34). In many cases, lysis systems contain additional regulatory proteins that inhibit or activate holin functions (19, 34, 38, 39). In the case of bacteriophage , it has been suggested that holin molecules accumulate and oligomerize in the CM throughout the period of late-gene expression and suddenly form lesions for endolysin passage (20).

Bacteriophage PRD1 is a broad-host-range phage that infects gram-negative bacterial species, such as Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa, that carry the phage receptor-encoding IncP-type multidrug resistance plasmid. PRD1 belongs to the Tectiviridae family, a group of icosahedral bacteriophages with a linear double-stranded DNA genome and an internal membrane component. The linear PRD1 genome contains covalently linked proteins at each 5' end that are involved in protein-primed replication by phage-encoded polymerase (7, 33). The PRD1 genome is surrounded by a membrane that follows the internal surface of the icosahedral capsid (1, 11). The external protein shell is composed of major capsid protein P3 trimers organized on a pseudo T = 25 lattice and bound together by protein P30 (1, 6, 32). The vertices, occupied by the spike-penton complex, are composed of proteins P2, P5, and P31 (2, 9, 25, 29). Protein P2 recognizes the receptor on the host cell surface (15, 37). After the virus binds to the receptor, an opening forms at one of the capsid vertices and the membrane is transformed into a tubular tail-like structure that penetrates the cell envelope for DNA delivery into the host cell (12, 16, 29). Virion-associated lytic enzymes are responsible for penetration of the peptidoglycan layer during DNA delivery into the cell (30).

In analysis of phage nonsense mutants, two PRD1 genes, XV and XXXV, have been shown to be involved in host cell lysis (25, 28). The product of gene XV, protein P15, is a soluble ß-1,4-N-acetylmuramidase that effectively degrades the peptidoglycan of the host cell, causing lysis (8). The gene XXXV product, protein P35, appeared to be a holin protein, most likely belonging to the class I lambdoid-type holins. PRD1-infected cells lyse prematurely upon the addition of cyanide, giving support to the idea of the presence of at least a two-component lysis system (28).

We further analyzed the PRD1 lysis system, expanding the examination of the premature lysis effect by applying metabolic inhibitors and evaluating the energy state of the cell. The efflux of intracellular K⁺ ions indicates an increase in the permeability of the CM, and the accumulation of the lipophilic cation tetraphenylphosphonium (TPP⁺) can be used to monitor the change in membrane voltage (). It was shown previously that during DNA delivery, PRD1 does not depolarize the host CM but induces a temporal K⁺ leakage from the cell, leading to increased outer membrane permeability to lipophilic compounds (12). Results obtained here elucidate the physiological changes that take place late in the infection due to holin action.

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Bacteria, bacteriophages, and plasmids. Bacterial strains, plasmids, and phages used in this study are listed in Table 1. Cells were grown at 37°C in Luria-Bertani (LB) medium (31), and when appropriate, ampicillin (150 µg/ml), tetracycline (20 µg/ml), chloramphenicol (25 µg/ml), and/or kanamycin (25 µg/ml) was added. Cell growth and lysis profiles were monitored by measuring the A₅₅₀ using a Selecta Clormic digital colorimeter (J. P. Selecta).

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TABLE 1. Bacteria, phages, and plasmids

Wild-type or mutant sus715 and sus232 phage agar stocks were prepared on Salmonella enterica DS88 and suppressor strains, respectively, as described elsewhere (28) (Table 1).

DNA manipulations. For the construction of plasmid pGZ9, the genomic region of PRD1 encoding protein P35 was amplified by PCR and inserted between the BamHI and HindIII sites of the pET24 vector. Plasmid pGZ9 was used for high-level expression of the holin protein. Plasmid pGZ15 was constructed by inserting the same PCR product between the BamHI and PstI sites of the pGZ119 vector and was used in the complementation assay. Plasmid pMG118 was constructed by amplifying the genomic region of PRD1 encoding protein P15 and inserting it between the EcoRI and HindIII sites of the pSU18 vector. DNA manipulations were performed using standard molecular cloning techniques (31). The nucleotide sequences of the inserts were determined by sequence analysis at the DNA Sequencing Laboratory, Institute of Biotechnology, University of Helsinki.

Electrochemical measurements. All measurements were carried out in thermostatic vessels at 37°C with aeration. Cells used for measurements were grown overnight at 37°C with aeration in the presence of antibiotics, diluted 35-fold with fresh LB medium, and grown to an A₅₅₀ of 1 (1 x 10⁹ CFU/ml). The cultures were infected with the phage, or in the case of the recombinant plasmids, gene expression was induced with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). Samples were withdrawn for electrochemical measurements at appropriate time points.

To measure the K⁺ gradient on the CM, cells were permeabilized using 40 µg/ml of polymyxin B sulfate (Sigma) and 5 µg/ml gramicidin D (Sigma). Calibration of the K⁺ electrode was carried out by adding a standard amount of KCl at the end of each measurement. For determination of the , the lipophilic cation TPP⁺ was used. To induce accumulation of TPP⁺, the outer membranes of the host cells were permeabilized using 200 µg/ml of polymyxin B. The amount of TPP⁺ that accumulated in a -dependent way was determined by measuring the release of TPP⁺ after addition of gramicidin D. Calibration of the measuring system using a standard amount of TPP⁺ chloride (Aldrich) was carried out at the end of each measurement. The concentrations of K⁺ and TPP⁺ ions in the medium were monitored by selective electrodes connected to an electrode potential amplifying system based on an ultralow-input bias current operational amplifier (model AD549JH; Analog Devices). The K⁺-selective electrode was purchased from Orion Research, Inc. (model 93-19). The Ag-AgCl reference electrodes (Orion Research, Inc.; model 9001) were indirectly connected to the vessels through agar-salt bridges. The construction and characteristics of the TPP⁺-selective electrode have been described previously (17). The values were calculated from a modified Nernst equation, as described elsewhere (12).

ATP measurements. The ATP content of cells was determined using the ATP biomass kit (BioThema). The total amount of ATP was measured by mixing 10 µl of cell suspension with 50 µl of extractant and adding 400 µl of ATP reagent. The amount of free ATP in the medium was determined by mixing 10 µl of cell suspension with 400 µl of ATP reagent. The ATP levels in both types of experiments were calibrated using a 1 µM ATP solution at the end of each measurement. The amount of light produced was measured with a model 1250 luminometer (LKB-Wallac).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteriophage PRD1 uses a two-component lysis system that is most effectively triggered by arsenate. In many cases the addition of energy-depleting agents like cyanide or dinitrophenol causes premature lysis of phage-infected cells (14, 20). Lysis is dependent on two phage-specific proteins, endolysin and holin. To further elucidate the mechanism involved, we investigated the effects of four different metabolic inhibitors.

It was previously shown that the addition of cyanide (blocking respiration due to the inhibition of cytochrome oxidase) to PRD1-infected Salmonella enterica cells at 35 min postinfection (p.i.) causes premature lysis (28) (Fig. 1A). We observed here that premature lysis was also induced using arsenate, an ATP-depleting agent (Fig. 1A). In spite of the ATP depletion, cells maintained a high K⁺ gradient, indicating the maintenance of CM integrity (12) (Table 2). NaN₃ inhibits bacterial growth by blocking respiration and oxidative phosphorylation due to inhibition of cytochrome oxidase and membrane H⁺-ATPase. The NaN₃-induced lysis was delayed and incomplete (Fig. 1A). NaF, an inhibitor of the enolase reaction and therefore of ATP formation using glycolytic substrates, showed only a weak lysis-inducing effect (Fig. 1A). This observation suggests that in LB medium and with intensive aeration, glycolysis plays a minor role in the conversion of energy in PRD1-infected cells.

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FIG. 1. (A) Effect of metabolic inhibitors on PRD1-infected Salmonella enterica DS88 cells. Inhibitors (20 mM final concentration) were added to the cell cultures at 35 min p.i. (indicated by the arrow). The figure shows noninfected cells with no poisons added (•), infected cells with no poisons added (), infected cells with sodium arsenate added (), infected cells with KCN added (), infected cells with NaF added (), and infected cells with NaN3 added (). In all cases a multiplicity of infection (MOI) of 10 was used. (B) Effect of arsenate on DS88 cells infected with wt PRD1 or lysis-defective mutants. Sodium arsenate (20 mM final concentration) was added at 35 min p.i. (indicated by the arrow). The figure shows infected cells (wt) with no arsenate added (), infected cells (wt) with arsenate added (•), holin mutant (sus715)-infected cells with no arsenate added (), sus715 mutant-infected cells with arsenate added (), endolysin sus232 mutant-infected cells with no arsenate added (), and sus232 mutant-infected cells with arsenate added (). In all cases an MOI of 10 was used.

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TABLE 2. Effects of metabolic inhibitors and PRD1 infection on the ATP content and membrane voltage of S. enterica DS88 cells

The arsenate effect was studied further using lysis-deficient mutants. Nonsuppressor DS88 cells infected with the PRD1 sus715 holin mutant did not show any signs of lysis, with or without addition of arsenate, at any time point (Fig. 1B). The same result was observed in the case of the endolysin sus232 mutant. However, in both mutant infections, host cell growth inhibition was observed, as in the case of the noninfected cells (Fig. 1B). If appropriate suppressor hosts were used, premature lysis was observed (data not shown). These observations confirm that both the phage holin and endolysin proteins are necessary for premature lysis.

Physiological changes during PRD1 infection. The function of holin is to permeabilize the CM for endolysin passage. The CM is a barrier for ions and other small hydrophilic molecules. Holins disintegrate this barrier and should allow nonspecific-ion and small-molecule movement across the CM, subsequently dissipating the ion gradients and the . We used ion-selective electrodes to analyze holin functions. K⁺ leakage could be detected as early as 40 min p.i. (Fig. 2A). This time point well precedes normal lysis (Fig. 2A). In contrast, no K⁺ leakage was observed with the holin mutant (sus715) infection or with the noninfected control (Fig. 2B and C). during infection was followed using a TPP⁺-selective electrode, as the distribution of TPP⁺ between the cell cytosol and the medium is a -dependent process. A considerable decrease in intracellular TPP⁺ was detected at about 55 min p.i. (Fig. 2A), about 10 min before the decrease in the cell culture turbidity was first observed. No decrease in intracellular TPP⁺ was observed in the case of infection with the holin mutant or with noninfected cells (Fig. 2B and C). Electrochemical data obtained from PRD1 infection indicated that holin incorporates into the CM and increases permeability to K⁺ approximately 15 min before the membrane voltage starts to dissipate.

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FIG. 2. Electrochemical assays of infected and noninfected S. enterica DS88 cells. (A) Cells infected with wt PRD1. (B) Noninfected cells. (C) Cells infected with the holin sus715 mutant. Cells were infected at an MOI of 25. •, A550; , amount of intracellular K+; , amount of accumulated TPP+.

The effects of metabolic inhibitors on intracellular ATP concentration and the of Salmonella enterica cells growing in LB medium were studied (Table 2). The addition of arsenate caused a drastic decrease in ATP but did not affect . Cyanide strongly affected both the and ATP levels. The effects of fluoride and azide were much weaker. These observations are consistent with the premature lysis results shown in Fig. 1A. The arsenate effect indicates that the most important lysis trigger could be the depletion of intracellular ATP, since the other poisons that affect the ATP pool (blocking respiration and/or inhibiting membrane H⁺-ATPase) resulted in a delayed and incomplete lysis.

The level of ATP in the infected cells was rather stable during the first 30 min of infection, followed by an 25% decrease reached at 40 min p.i. This level of ATP stayed reasonably stable for the next 20 min of infection (Fig. 3B). The second decrease in the total level of ATP started 60 min p.i., following the decrease of optical density (Fig. 3A). The level of free ATP in the medium was low (around 1 to 2% of the total ATP) during the first 60 min of the infection and started to increase about 5 min prior to cell lysis (Fig. 3B, inset). However, no increase in the level of ATP in the medium was registered during the period of 30 to 40 min p.i., when the drop in the total amount of ATP in the infected cells occurred (Fig. 3B).

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FIG. 3. ATP content of PRD1-infected S. enterica DS88 cells. Both A550 (A) and ATP content (B) were measured from the same samples. •, total ATP content; , amount of free ATP in the growth medium. The cells were infected at an MOI of 10 at time point zero.

At elevated temperatures, PRD1-infected groE mutant temperature-sensitive host cells were lysis defective. We studied the lysis phenomenon of PRD1-infected groE mutant host cells, as the translocation and membrane insertion of holin proteins may be associated with chaperonin activity (5, 13, 22). E. coli strains carrying mutations in groEL or groES were tested for temperature sensitivity. All strains grew normally at 37 and 40°C; however, at 42°C, the mutant strains showed clear growth inhibition (not shown) but the wild-type (wt) strain was not affected. Mutant strains infected with wt PRD1 showed a clear lysis phenotype at 37°C (Fig. 4A). In contrast, at 40°C, the mutant strains showed no signs of lysis (Fig. 4B) whereas the wt strain lysed normally.

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FIG. 4. Lysis profiles of E. coli strains carrying mutations in the groEL or groES genes infected with wt PRD1 at 37°C (A) and at 40°C (B). •, wt strain DW720(pLM2); , groEL mutant strain DW721(pLM2); , groES mutant DW719(pLM2); , groEL mutant DW717(pLM2); , groEL mutant DW716(pLM2). Cells were infected at an MOI of 30 at time point zero.

Cloning of the holin and endolysin genes. To enable specific measurements of the effects of the holin and endolysin proteins on the CM, the corresponding genes were cloned into plasmid vectors. To allow the simultaneous expression of holin and endolysin in the same cell, the corresponding genes were cloned into two different plasmids with compatible replicons (Table 1). The holin expression construct, pGZ9, was under the control of the T7 promoter, and the endolysin construct, pMG118, was under the control of the lac promoter. The holin gene was also cloned under the control of the tac promoter (pGZ15) for complementation assays.

The functionality of the recombinant holin proteins was tested in a complementation assay. The titers of the PRD1 sus715 phage with a mutation in the holin gene were determined on E. coli strains carrying the different recombinant holin constructs. As a control, the titers were determined on three different suppressor hosts. The mutation was suppressed in all strains tested, resulting in an almost-six-log increase in the titer (Table 3). However, the different strains all showed different plaque morphologies. The background titer (representing reversions of the mutation and ribosomal read-through of the stop codon) was determined on a nonsuppressor strain containing the cloning vector (backbone) only. Surprisingly, all control strains except that containing pSU18 showed considerable increases in sus715 phage titer when IPTG was added to induce recombinant-protein expression. These observations limited us to consider only the experiments under "noninduced" conditions, where the holin gene (pGZ15) complemented the defect in sus715 (Table 3).

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TABLE 3. Functionality of the different holin gene constructs tested by complementation assay

Effects of plasmid-borne PRD1 holin and endolysin genes on cell physiology. The physiological effects were tested on HMS174(DE3) cells expressing either holin (pGZ9), endolysin (pMG118), or both of these proteins. The same cells containing the corresponding cloning vectors without inserts were used as controls. After induction of holin gene expression, K⁺ leakage started 75 min postinduction, the accumulation of TPP⁺ decreased starting at 90 min, and cell growth stopped approximately 100 min postinduction, but no lysis was detected (Fig. 5A). In contrast, the control cells did not stop growing, did not leak K⁺, and showed no decrease in the amount of accumulated TPP⁺ (Fig. 5B). Similarly, the strain expressing the cloned endolysin gene showed no K⁺ leakage, growth inhibition, or decrease in the accumulation of TPP⁺ (Fig. 5C and D). When the holin and endolysin genes were coexpressed in the same cell, K⁺ leakage was detected 50 min postinduction, reduced accumulation of TPP⁺ was observed at about 90 min, and a decrease of turbidity started at approximately 100 min postinduction (Fig. 5E). None of these effects could be detected in the control strain (Fig. 5F). The data on electrochemical properties of the cells carrying recombinant proteins revealed that the K⁺ leakage and loss of were functions of the holin protein and that endolysin was needed for cell lysis.

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FIG. 5. Electrochemical assays of recombinant E. coli cells containing holin and/or endolysin genes. (A) HMS174(DE3)(pGZ9) cells containing the holin gene; (B) HMS174(DE3)(pET24) control strain; (C) HMS174(DE3)(pMG118) cells containing the endolysin gene; (D) HMS174(DE3)(pSU18) control strain; (E) HMS174(DE3)(pGZ9)(pMG118) cells containing the holin and endolysin genes; (F) HMS174(DE3)(pET24)(pSU18) control strain. •, A550; , intracellular amount of K+; , amount of accumulated TPP+. The expression of genes was induced using 1 mM IPTG at time point zero.

Surprisingly, IPTG induced an increase in the level of ATP in the cells coexpressing holin and endolysin genes, as well as in the control cells (Fig. 6B). This effect was observed starting 25 min postinduction, and at 60 min, the intracellular ATP content reached the highest level, followed by a decrease. A faster decrease in ATP content in the cells coexpressing holin and endolysin proteins was registered at 100 min postinduction than in the control cells (Fig. 6A). At 150 min postinduction, the content of ATP in the vector-containing cells returned to the preinduction level, but it continued to drop in the case of cells coexpressing the holin and endolysin genes.

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FIG. 6. ATP content of recombinant E. coli cells with and without holin and endolysin genes. (A) A550 and (B) the total amount of ATP were measured from the same samples. •, induced HMS174(DE3)(pGZ9)(pMG118) cells containing the holin and endolysin genes; , noninduced HMS174(DE3)(pGZ9)(pMG118) cells; , induced HMS174(DE3)(pET24)(pSU18) control cells. The expression of genes was induced using 1 mM IPTG at time point zero.

Cells carrying recombinant lysis proteins showed immediate arsenate-inducible lysis. Since the simultaneous expression of the recombinant holin and endolysin genes had effects in the cells qualitatively similar to those of the wild-type virus infection, lysis induction with arsenate was tested. The induction of recombinant protein expression caused either growth inhibition (in cells containing either endolysin or holin) or lysis (in cells containing both endolysin and holin) (Fig. 7). When arsenate was added to the cells, a delay or a complete lack of lysis was detected in the case of single-protein expression. However, when both the holin and endolysin proteins were present, clear lysis was observed. In these time-dependent experiments, the earliest time point for arsenate-induced lysis occurred approximately 50 min postinduction (data not shown).

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FIG. 7. Effect of arsenate on recombinant E. coli cells. Gene expression was induced using 1 mM IPTG at time point zero. •, HMS174(DE3)(pGZ9) cells containing the holin gene and no arsenate; , HMS174(DE3)(pGZ9) cells with arsenate added; , HMS174(DE3)(pMG118) cells containing the endolysin gene with no arsenate; , HMS174(DE3)(pMG118) cells with arsenate added; , HMS174(DE3)(pGZ9)(pMG118) cells containing the holin and endolysin genes with no arsenate; , HMS174(DE3)(pGZ9)(pMG118) cells with arsenate added. Twenty millimolar sodium arsenate was added at 50 min postinduction (indicated by the arrow).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The simultaneous presence of both holin and endolysin is the prerequisite for cellular lysis during the normal PRD1 life cycle. The same phenomenon was observed when these proteins were expressed as recombinant products (Fig. 2 and 5). In addition, premature lysis, induced by metabolic inhibitors, requires the presence of both proteins exclusively (Fig. 7). These results confirm that the PRD1 lysis system resembles those operating in a number of other phages (34, 38, 39, 40).

Infected cells became sensitive to premature lysis approximately 35 min postinfection, or about 20 min prior to normal lysis (Fig. 1A). This phenomenon could be correlated most closely with the depletion of the cellular ATP pool because arsenate, which depletes ATP, caused immediate lysis, while other agents affecting ATP synthesis had a delayed effect. Holin-dependent leakage of intracellular K⁺ also began at approximately 35 min p.i. (Fig. 2A), indicating that enough holin molecules had localized to the CM to allow leakage of K⁺ along its concentration gradient. However, in spite of the potential to lyse, as shown by arsenate-induced lysis, (Fig. 1A and B), the lysis function of infected cells is repressed. It is noteworthy that at this time point of infection, the is not yet affected. Taking these observations together, it is apparent that there is an ATP-associated control mechanism that prevents the transformation of holin complexes into an endolysin-permeable form.

It is known that GroEL/GroES chaperonins assist in the insertion of PRD1 proteins into the virion membrane (21, 22). These chaperonins also translocate several other integral membrane proteins to the CM (5, 13) in a process that is induced by a low cellular ATP concentration. It is conceivable that chaperonins could be involved in the storage of holin molecules and in their delivery into the CM, but these possibilities need to be proven by further studies. The recombinant system studied here was similar to normal infection, in that lysis occurred only in the presence of both the holin and endolysin proteins and K⁺ leakage was dependent solely on the presence of the holin protein (Fig. 5A and 7). However, the decrease in turbidity and leakage of K⁺ occurred much more slowly than with the native virus infection. The possibility that some additional elements encoded by the virus modulate the lysis events or that the production of the recombinant proteins is low cannot be excluded.

We also observed the intriguing phenomenon of a strongly increased background in complementation experiments when IPTG was added, in particular with backbone vectors encoding ampicillin or kanamycin resistance. This observation emphasizes the need for proper controls when very sensitive assays are used. However, we did not observe such ambiguities when the recombinant proteins were expressed from plasmid pET24 (Km^r) for electrochemical measurements (Fig. 5A and E and 6).

We have studied the physiological effects of the PRD1 lysis system using infected cells and cells expressing both the recombinant holin and endolysin proteins. Our results are in agreement with the model proposed previously (35), where there are two different holin complexes with altering gating properties. The translocation of endolysin is an obvious function for the holin protein. However, the drop in ATP (caused possibly by viral macromolecular synthesis and packaging) seems to be the initiating event leading to lysis, as indicated by the premature lysis induced by ATP depletion. It is likely that the lysis occurs through the lowering of the level mediated by K⁺ leakage and possibly via delivery of GroEL-associated holins to the CM. This is supported by the observation (Fig. 4) that PRD1-infected groE temperature-sensitive mutant host cells did not lyse at elevated temperatures. The lysis events may be controlled both at the level of gene expression (time and magnitude of expression) and posttranslationally by the regulation of electrolyte fluxes once the primary holin complex is assembled.

 
We thank Marika Grahn for providing plasmid pMG118.

This work was supported by research grants from the Academy of Finland (1202108, 1202855 [D.H.B.] [Finnish Center of Excellence Program 2000-2005], and 1201964 [J.K.H.B.]). G.Z. was supported by the European Community SOCRATES/ERASMUS program. R.D. was a Lithuanian State Fellowship holder.

 
* Corresponding author. Mailing address: Biocenter 2, P.O. Box 56 (Viikinkaari 5), FIN-00014 University of Helsinki, Finland. Phone: 358 9 191 59100. Fax: 358 9 191 59098. E-mail: dennis.bamford{at}helsinki.fi.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, August 2005, p. 5397-5405, Vol. 187, No. 15
0021-9193/05/$08.00+0     doi:10.1128/JB.187.15.5397-5405.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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