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Journal of Bacteriology, May 2005, p. 3521-3527, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3521-3527.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Linear Double-Stranded DNA of Phage Bam35 Enters Lysogenic Host Cells, but the Late Phage Functions Are Suppressed

Aura Gaidelyt,^1,2 Silja T. Jaatinen,¹ 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 13 December 2004/ Accepted 14 February 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bam35, a temperate double-stranded DNA bacteriophage with a 15-kb linear genome, infects gram-positive Bacillus thuringiensis cells. Bam35 morphology and genome organization resemble those of PRD1, a lytic phage infecting gram-negative bacteria. Bam35 and PRD1 have an outer protein coat surrounding a membrane that encloses the viral DNA. We used electrochemical methods to investigate physiological changes of the lysogenic and nonlysogenic hosts during Bam35 DNA entry and host cell lysis. During viral DNA entry, there was an early temporal decrease of membrane voltage associated with K⁺ efflux that took place when either lysogenic or nonlysogenic hosts were infected. Approximately 40 min postinfection, a second strong K⁺ efflux was registered that was proposed to be associated with the insertion of holin molecules into the plasma membrane. This phenomenon occurred only when nonlysogenic cells were infected. Lysogenic hosts rarely were observed entering the lytic cycle as demonstrated by thin-section electron microscopy.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Temperate double-stranded DNA bacteriophages may enter either the lytic pathway that leads to host cell death or the lysogenic pathway in which the phage genome is maintained and normal cell growth continues. Most temperate phages studied enter long-term relationships with their hosts by integrating their genome into the host chromosome. In addition, some phages reside as nonintegrated circular or linear plasmids in their host cells, thereby establishing a lysogenic state (for reviews, see references 8 to 10). In the prophage state, while most viral genes are silent, those that are expressed are implicated in the control and maintenance of lysogeny. Many temperate bacteriophages encode -C_I repressor-like proteins that maintain the prophage state and also may prevent the propagation of superinfecting phages (repression/immunity function) (25). Another group of genes regulate entry exclusion phenomena (14, 38). Some prophage genes may also alter host properties such as ecological fitness, virulence of nonpathogenic host cells, and increased virulence of pathogenic hosts (6, 41). Lysogeny-related genes (encoding proteins such as integrase, excisionase, and repressors and antirepressors) are organized into lysogeny modules that are similar in temperate phages infecting evolutionarily related bacterial genera (24). Infective virions can be produced in cells containing an intact prophage genome. This induction can occur spontaneously in a small fraction of lysogenic cells. Alternatively, specific environmental signals can cause simultaneous induction of prophage in the majority of host cells. Bam35 can exist as a linear plasmid in some Bacillus thuringiensis strains, and it has been proposed to have a protein-primed replication system (37, 40). Linear plasmids of 15 kb are commonly carried in bacilli, and some have been shown to be related to Bam35 (19, 40).

Viruses have evolved mechanisms to enter the host cell without seriously interfering with cell membrane integrity or metabolic activity (for reviews, see references 22, 23, and 28). In contrast, cell lysis is the prevailing way to release newly assembled viruses from the bacterial cell. The lysis system, composed of endolysin and holin, is the most common mechanism for progeny release employed by bacteriophages infecting both gram-positive and gram-negative hosts (26, 43). Viral endolysins accumulate in the host cell cytosol, whereas holins, being small integral membrane proteins, accumulate in the plasma membrane (PM) and, in a time-dependent manner, form openings allowing the endolysin to attack the cell peptidoglycan (5, 42, 44, 46).

The bacteriophage Bam35 infects the gram-positive bacterium B. thuringiensis serovar israelensis. Bam35, initially isolated and characterized in 1978 (2), belongs to the family Tectiviridae, which consists of several double-stranded DNA bacteriophages having a membrane beneath the icosahedral protein shell. Recently the genome of a clear-plaque mutant of Bam35 was sequenced and the virus was characterized in more detail (29). Bam35 and bacteriophage PRD1, the best-characterized member of the family Tectiviridae, have a similar genome size (15 kb) and organization but practically no detectable sequence similarity (29). As determined by negative-stain electron microscopy, Bam35 morphology closely resembles that of PRD1, despite the fact that these phages infect very different hosts (PRD1 infects gram-negative bacteria). Recently, this was confirmed by comparison of the high-resolution X-ray structure of PRD1 (1, 11) with the cryoelectron microscopy-based three-dimensional image reconstruction of Bam35 (P. A. Laurinmäki, J. T. Huiskonen, D. H. Bamford, and S. J. Butcher, unpublished data).

The phospholipid composition of Bam35 and PRD1 virions, as well as their respective hosts, was determined previously (21). It was shown that, in both cases, virion-associated phospholipids are host PM derived but selectively incorporated into the virion. PRD1 receptor recognition results in the formation of an opening at one of the capsid vertices (15, 34). Subsequently, part of the viral membrane is transformed into a tubular structure that crosses the capsid through the opening. The formed tail-like structure penetrates the cell envelope, and DNA is delivered into the host cytosol (13, 16). Similar tail tube structures have been observed in Bam35 (2), suggesting that the viral membrane also might be involved in delivery of the phage genome into the host cell. Two lytic enzymes, proteins P7 and P15, are connected to the PRD1 membrane and are involved in DNA entry (31). P15, with muramidase activity, also is responsible for host cell lysis (7, 32). Similarly, two lytic enzymes have been described in Bam35 and its close relative Gil01 (29, 39). The recent identification of PRD1 holin indicates that the two-component lysis system operates in PRD1 (33, 46); however, the holin gene has not yet been identified in Bam35.

In most cases, the virus-host cell interaction induces detectable changes in the outer membrane permeability and membrane voltage (). The efflux of intracellular K⁺ indicates increased permeability of the PM, and the accumulation of lipophilic cations (e.g., tetraphenylphosphonium [TPP⁺]) is used to measure in bacterial cells (12, 17, 45). PRD1 entry does not depolarize the host PM but induces a temporal K⁺ leakage from the cell. In addition, PRD1 infection increases the host outer membrane permeability to lipophilic compounds (13). Here, we studied the changes in and K⁺ gradient when lysogenic or nonlysogenic B. thuringiensis cells were infected with Bam35. We also provide evidence that an endolysin-holin system likely operates in the release of Bam35 virions from the host cell.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteria and phage cultivation. The bacterial strains used in this study are listed in Table 1. B. thuringiensis cells were cultivated in Luria-Bertani broth (LB) (35). Lysogenic or resistant cell lines were obtained by isolating microcolonies from the centers of plaques and by isolating colonies from confluently lysed plates of B. thuringiensis serovar israelensis HER1410 infected with a clear-plaque variant of Bam35 (29). Remaining free phage particles were eliminated through eight consecutive single-colony purification steps. The presence of Bam35 DNA in the cells was verified both by isolating total DNA from bacterial cells (Wizard genomic DNA purification kit; Promega) and by PCR on DNA from single bacterial colonies (20) with specific primers hybridizing to the ends of Bam35 ORF18 encoding the major coat protein (29).

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TABLE 1. B. thuringiensis strains used in this study

Lysogenization and resistance frequencies. HER1410 cells (2 x 10⁸) in a liquid culture were infected (multiplicity of infection [MOI], 3 or 30), incubated with aeration at 37°C for 10 min, and plated. Survivors (from 1/40 of the infection mixture) were isolated and tested for their ability to form plaques and to release virions spontaneously and for the presence of the 15-kb DNA Bam35 genome.

Cell flocculation assay. To test cell flocculation, cells from a single colony were grown overnight at 37°C with aeration, followed by incubation at room temperature for approximately 20 h without aeration. The flocculation level was estimated visually.

Detection of spontaneously released virions. To detect spontaneously released viruses, cells were diluted to 5 x 10⁷ CFU/ml and grown at 37°C for 3 h. The supernatants of the cell cultures were plated on HER1410 lawns.

Phage adsorption test. The phage adsorption test was performed by mixing 300 to 600 phage particles with 1.2 x 10⁷ cells grown to a cell density of 2 x 10⁸ CFU/ml. The infection mixture was incubated for 10 min at 37°C with aeration, and the cells were removed by centrifugation (microcentrifuge at 12,000 rpm for 3 min at 22°C), followed by washing with LB. The number of nonadsorbed phage particles was determined by plating the supernatants on HER1410 lawns.

Evaluation of premature cell lysis. To test the premature lysis of infected cells, the cells were grown at 37°C to 2 x 10⁸ CFU/ml and infected with Bam35 using an MOI of 20. At different time points postinfection, a metabolic inhibitor (sodium azide, sodium fluoride, sodium arsenate, or potassium cyanide) was added to a final concentration of 20 mM. The culture turbidity was followed using a Klett-Summerson colorimeter (A₅₄₀).

Measurements of ion fluxes. Ion flux measurements were performed as described previously (13), with the following modifications. The experiments were carried out simultaneously in two 5-ml thermostat-regulated (37°C) vessels with aeration. The concentrations of K⁺ and TPP⁺ ions in the medium were monitored by selective electrodes. To describe the early changes in PM permeability of Bam35-infected cells, the cells were grown to 2 x 10⁸ CFU/ml, collected by centrifugation, resuspended in LB (pH 8.0; pH adjusted with NaOH) to obtain 1/50 of the original volume, and kept on ice until used (maximum, 4 h). The concentrated cell suspension (200 µl) was added to 5 ml LB (for TPP⁺ measurements, the LB contained 3 µM TPP⁺), incubated for 5 min, and infected with freshly made Bam35 stock. The electrodes were calibrated by adding an appropriate amount of TPP⁺ or K⁺ at the beginning (TPP⁺) or at the end (K⁺) of every experiment. To determine the amount of intracellular K⁺ and TPP⁺, the PM was permeabilized by the addition of gramicidin D (GD) (4 µg/ml).

To describe the late changes in PM permeability of Bam35-infected cells, 400 ml of the bacterial culture (grown to 2 x 10⁸ CFU/ml at 37°C) was infected with Bam35 (MOI, 10) and incubated at 37°C with aeration. At each time point, two 5-ml samples were taken and transferred into the vessels. TPP⁺ was added to a final concentration of 3 µM (in TPP⁺ measurements only) and, after stabilization of the electrode potential, GD was added. Both K⁺ and TPP⁺ electrodes were calibrated at the end of every experiment. The K⁺ concentration in LB was estimated from calibration curves.

Characteristics of TPP⁺-selective electrodes have been described previously (13, 17). The electrodes were connected to the electrode potential-amplifying system, based on an ultralow input bias current operational amplifier (AD549JH; Analog Devices). The amplifying system was connected to a computer through the data acquisition board AD302 (Data Translation, Inc., Marlboro, Mass.). TPP⁺ chloride and GD were purchased from Sigma.

Electron microscopy. For thin-section electron microscopy, B. thuringiensis cells were grown in LB at 37°C to a density of 2 x 10⁸ CFU/ml and infected with Bam35 using an MOI of 12. Samples were collected at different time points after infection and fixed with 3% (vol/vol) glutaraldehyde. As a control, noninfected bacteria were fixed at a cell density of 4 x 10⁸ CFU/ml. After 20 min of fixation at room temperature, cells were collected, washed twice with 20 mM potassium phosphate buffer (pH 7.2), and prepared for transmission electron microscopy as previously described (3). The electron micrographs were taken with a JEOL 1200 EXII electron microscope (at the EM unit, Institute of Biotechnology, University of Helsinki) operating at 60 kV.

DNA sequencing. The DNA for sequencing was amplified by PCR using Bam35-specific primers and native Pfu polymerase (MBI Fermentas). The DNA templates for amplification reactions were prepared from colonies and plaques as described elsewhere (18, 20). PCR products (about 1.8 kb) were purified using microcentrifuge spin columns (QIAquick PCR Purification Kit; QIAGEN), and both strands of the DNA were sequenced (Sequencing Laboratory, Institute of Biotechnology, University of Helsinki).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Alternative hosts for Bam35. Bam35 is a Bacillus-specific phage and can lyse Bacillus megaterium strain Tiberius and some strains of Bacillus cereus and B. thuringiensis (2). In order to test the sensitivity of B. thuringiensis strains other than HER1410 to Bam35, we used a plaque assay to screen 14 strains obtained from the Bacillus Genetic Stock Center. Only one alternative host strain in addition to HER1410 was found. B. thuringiensis serovar kurstaki 4D22 was able to support Bam35 propagation with the same plating efficiency as HER1410 but gave more turbid plaques. In liquid cultures, the titer of Bam35-infected 4D22 cell lysates was about 35% of that of HER1410. In addition, there were differences in Bam35 adsorption to HER1410 and 4D22 hosts (Table 2).

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TABLE 2. Characteristics of B. thuringiensis strains used in this study

Bam35-resistant and lysogenic HER1410 cells. Liquid culture cells surviving Bam35 infection were isolated. Plaque assay, phage adsorption, and spontaneous plaque production tests were carried out. Bam35-resistant and lysogenic HER1410 colonies appeared with approximately similar frequencies (Table 3). Three cell lines (HER1410_R19, HER1410_L3, and HER1410_L5) were chosen for further analyses (Table 2).HER1410_R19 did not support virus propagation or produce virus particles spontaneously and did not adsorb Bam35 particles. PCR from single colonies showed no Bam35-specific DNA. Consequently, HER1410_R19 is a genuine Bam35-resistant strain. HER1410_L3 and HER1410_L5 did not support virus propagation (plaque assays) but adsorbed Bam35 with high efficiency (Table 2) and released small amounts of virus into culture supernatants. PCR from single colonies and the sequence analysis revealed Bam35-specific DNA in these cells. Purified DNA from HER1410_L3 and HER1410_L5 strains contained 15-kb DNA elements that were not found in the original (HER1410) or resistant strains (not shown, but see previous work, where this phenomenon is described for HER1410_L5 strain [37]). Consequently, HER1410_L3 and HER1410_L5 are lysogenic strains carrying Bam35 prophages.

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TABLE 3. Frequencies of HER1410 lysogenization and mutation to resistance

Bam35 entry induces depolarization of the PM. Concentration of the indicator TPP⁺ in the medium was registered during the first 10 min of Bam35 infection (Fig. 1). Rapid release of HER1410 cell-accumulated TPP⁺ (PM depolarization) began 1.5 to 2 min after phage addition and was followed by a slow uptake of TPP⁺ (PM repolarization) starting 4 to 5 min postinfection (Fig. 1A). Interestingly, the same time course of TPP⁺ ion movements was observed when lysogenic HER1410_L3 cells were infected. In the case of serovar kurstaki (4D22), Bam35 induced slow leakage of accumulated TPP⁺ that began about 1.5 to 2 min postinfection but subsequent PM repolarization was not observed (Fig. 1B). The time course of TPP⁺ accumulation in nonadsorbing derivative cells (HER1410_R19) infected with Bam35 was the same as in the case of noninfected HER1410 cells. The initial drop in TPP⁺ concentration in this experiment was due to TPP⁺ binding by the input virus and the increased volume of infection mixture (insert in Fig. 1A). The phage-induced efflux of accumulated TPP⁺ was MOI dependent (Fig. 1C).

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FIG. 1. Effects of Bam35 infection on TPP+ (A and C) and K+ (B and D) ion fluxes across the PM of different B. thuringiensis strains and cell lines during the first 10 min of infection. The experiments were carried out at 37°C in LB (pH 8.0) containing 3 µM TPP+ (in panels A and C). Approximately 4 x 108 CFU/ml cells were infected with Bam35 using an MOI of 20 (A and B) or as indicated below (C and D). GD was added to a final concentration of 4 µg/ml. Experiments were done with B. thuringiensis strains HER1410 (black, infected; yellow, noninfected) and 4D22 (blue) and cell lines HER1410_L3 (red) and HER1410_R19 (green) (A and B). HER1410 cells were infected with Bam35 using MOIs of 16 (red), 4 (blue), and 2 (green) (C and D). The insert in panel A indicates the decrease in TPP+ concentration due to TPP+ binding and dilution by the added phage suspension.

Bam35 entry induces K⁺ leakage. The addition of Bam35 particles to the wild-type (HER1410) and lysogenic (HER1410_L3) cell suspensions resulted in the rapid efflux of K⁺ ions starting approximately 2 min postinfection and lasting approximately 2 min (Fig. 1B). The magnitude of phage-induced K⁺ leakage from 4D22 cells was about one-third of that observed with HER1410 cells, and the time course was longer (approximately 3.5 min). There was neither a significant change in K⁺ concentration in the medium after addition of phage particles to the resistant (HER1410_R19) cells nor a spontaneous leakage of K⁺ from noninfected HER1410 cells detected. The magnitude of the phage-induced K⁺ efflux was consistent with the phage effect on of each host strain. As in the case of TPP⁺ accumulation, the magnitude of K⁺ leakage was MOI dependent (Fig. 1D).

Late physiological changes in Bam35-infected cells. Bam35-induced changes in PM permeability late in the infection cycle were followed by sampling throughout one-step growth experiments. An increase of TPP⁺ accumulation by infected HER1410 cells was detected during the first half of the infection cycle. Accumulation of K⁺ by infected cells also was observed during the first 28 min of infection (Fig. 2A). Strong efflux of K⁺ from infected cells started about 20 min before any decrease in the turbidity of the cell suspension was detected (50 min postinfection), and there was no measurable K⁺ gradient on the PM at the time of lysis. In addition, when the K⁺ leakage began, (or TPP⁺ accumulation) dropped rapidly and remained at approximately half of the initial level at the time of cell lysis. As a control, increases of TPP⁺ and K⁺ accumulation by noninfected HER1410 cells were observed due to the growth of the culture (Fig. 2B). The addition of Bam35 particles to lysogenic (HER1410_L3) cells did not result in cell lysis or cause measurable late K⁺ leakage or a decrease in TPP⁺ accumulation (Fig. 2C). However, Bam35 entry induced early depolarization of the PM and the early efflux of K⁺ (Fig. 1A and B). Electrochemical parameters of noninfected lysogenic cells (Fig. 2D) did not differ considerably from those of the infected cells. This result shows that the majority of infected lysogenic cells do not lyse and consequently do not release progeny viruses.

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FIG. 2. Changes in B. thuringiensis HER1410 and HER1410_L3 cell energetics during Bam35 infection. The cells were grown in a large volume (400 ml) and infected at time zero (MOI, 10). Two 5-ml samples of cell suspension were withdrawn at each time point, and the amounts of intracellular K+ and accumulated TPP+ were measured (see Materials and Methods). Infected HER1410 cells, A; noninfected HER1410 cells, B; infected HER1410_L3 cells, C; noninfected HER1410_L3 cells, D. Symbols: K+, closed circles; TPP+, open triangles; and OD, open circles.

Lysogenic cells produce viruses sporadically. Bam35-infected and noninfected lysogenic cells were subjected to thin-section electron microscopy. Electron micrographs (Fig. 3) of Bam35-infected lysogenic (HER1410_L3) cells revealed both empty and filled virus particles associated with the cell surface, confirming the unaltered adsorption (Table 2). No virus particles were observed on the surface of HER1410_R19 cells. This indicates that there are no active receptors on the resistant cell surface. Occasional lysing cells were observed when infected lysogenic bacteria (HER1410_L3) were examined. The titer of infected lysogenic culture supernatants was low. However, the number of virus particles produced per induced cell did not deviate considerably from normal infection (Fig. 3 C and E), as observed using electron microscopy. Interestingly, rare lysis events of noninfected lysogenic (HER1410_L3) cells producing only a few virus particles were also captured. These observations were consistent with the results obtained with plaque assays.

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FIG. 3. Thin-section electron micrographs of Bam35-infected (A, C, D, and E) or noninfected (B) B. thuringiensis cells. Lysogenic cell HER1410_L3 with surface-associated virus particles (A). Partially lysed noninfected HER1410_L3 cell; a few virus particles are visible in the cell interior (B). Progeny viruses observed in HER1410_L3 cell at 55 min postinfection (C). Resistant cell HER1410_R19 after Bam35 addition; no bound viruses are seen (D). Partially lysed HER1410 cell with progeny viruses 40 min postinfection (E). Black arrows mark DNA-containing virus particles, and white arrow points to an empty particle. Bar represents 200 nm in all panels.

Bam35 infection induces holin-type effects in the host cell. Premature lysis of infected cells caused by the addition of metabolic inhibitors has been proven to be a hallmark of the endolysin-holin lysis systems operating in many bacteriophage infections (Escherichia coli phage [4, 30], gram-negative bacterium-infecting phage PRD1 [33, 46], and Bacillus subtilis phage 29 [36]). We tested whether the addition of metabolic inhibitors to Bam35-infected cells could trigger premature lysis. Sodium azide (inhibitor of cytochrome oxidase and membrane H⁺-ATPase), potassium cyanide (inhibitor of cytochrome oxidase), or sodium arsenate (reduces the concentration of intracellular ATP by arsenolyzation of acetyl phosphates) inhibited growth but did not induce lysis of noninfected cells (shown for azide in Fig. 4). These poisons, added at the proper time, induced premature lysis of infected HER1410cells (Fig. 4A). Normally, Bam35-infected HER1410 cells lysed at approximately 50 min postinfection. Sodium fluoride, an inhibitor of the enolase reaction, did not inhibit growth of noninfected cells or induce premature lysis of infected cells. Timing experiments with sodium azide, sodium arsenate, or potassium cyanide revealed that these poisons induce premature lysis if added at 35 min postinfection or later (not shown). The addition of arsenate or azide to 4D22 cells at 40 min postinfection also resulted in premature lysis (not shown). Premature lysis inducers had no effect on both infected and noninfected resistant cells (HER1410_R19, shown for azide in Fig. 4B) other than growth inhibition. Growth inhibition of infected lysogenic (HER1410_L3 or HER1410_L5) cells could be detected at approximately 60 to 110 min postinfection. However, infected (as well as noninfected) HER1410_L3 and HER1410_L5 cells did not lyse after the addition of the metabolic inhibitor (shown for HER1410_L3 and azide in Fig. 4C).

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FIG. 4. Effect of metabolic inhibitors on infected or noninfected B. thuringiensis strains HER1410 (A), HER1410_R19 (B), and HER1410_L3 (C). Cells were grown to a cell density of 2 x 108 CFU/ml (37°C) and infected at time zero using an MOI of 20. Noninfected cells: no metabolic inhibitors added (closed circles) or NaN3 added (open circles). Bam35-infected cells: no metabolic inhibitors added (closed triangles), NaN3 added (open triangles), KCN added (closed squares), arsenate added (open squares), or NaF added (closed diamonds). The arrows indicate the time point of addition of inhibitors.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The morphology of the Bam35 virion, including the dynamic tail tube, is similar to that of PRD1 (29), a well-studied lytic virus infecting gram-negative bacteria. Bam35 infects gram-positive hosts and can establish a prophage state and reside as a linear plasmid in the cell (37, 40). Early events in PRD1 infection include a transient K⁺ efflux without depolarization of the PM (12, 16). In contrast, Bam35 infection caused, in addition to transient K⁺ efflux, a temporal decrease in (Fig. 1), as is often detected in phage infections (23). In both virus systems, the early changes in host cell permeability indicate local openings in the PM associated with the viral genome entry. Bam35 has to cross the gram-positive cell wall, which lacks the outer membrane and contains a thicker peptidoglycan layer compared to the gram-negative bacteria (hosts for PRD1). The internal osmotic pressure of gram-positive bacteria is higher than in gram-negative bacteria (27). Consequently, in order to decrease osmotic pressure inside the B. thuringiensis cell Bam35 has to induce a stronger K⁺ leakage and therefore to depolarize the PM. We assume that the tubular membrane structure of Bam35 might be involved in the PM penetration step similarly to the case of PRD1.

In this study, we obtained an alternative host for Bam35 (4D22) and generated lysogenic (HER1410_L3) and resistant (HER1410_R19) derivatives of HER1410 (Table 2). Weaker Bam35 entry-induced effects on 4D22 PM (Fig. 1) and the lower number of progeny viruses produced were, most probably, results of the less effective virus adsorption to these cells (Table 2). Interestingly, the lysogenic cells adsorbed Bam35 particles with the same efficiency as the nonlysogenic ones (HER1410). In addition, the entry-associated changes of PM permeability parameters (including their amplitudes) were the same as in the case of nonlysogenic cells but deviated from those of the resistant cells (Fig. 1 and 2). These observations indicate that the virus enters the lysogenic cells with the same efficiency as the nonlysogenic HER1410 cells. However, the infected lysogenic strains did not lyse but had a slower growth rate beginning at the time of normal lysis (Fig. 4C). Using electron microscopy, we observed occasional lysing cells (Fig. 3C). Their number could account for the reduced turbidity increase.

Starting at approximately 35 min postinfection, Bam35-infected cells become sensitive to premature lysis when metabolic inhibitors are administered (Fig. 4). This timing coincides with the efflux of K⁺ from infected cells (Fig. 2). Similar phenomena were observed in PRD1 infection and were associated with the endolysin-holin system responsible for cell lysis (46). This parallel suggests that an endolysin-holin system might operate also in Bam35 infection. Two genes encoding putative lytic enzymes have been observed in Bam35 or its close relative Gil01 (29, 39). The Bam35 holin gene, however, remains to be identified. The premature lysis was not detected for infected lysogenic cells (Fig. 4C). In addition, electrochemical measurements did not show any characteristics specific for the presence of the holin proteins in the membranes of these cells (K⁺ efflux, decrease of , Fig. 2). This suggests that virus production in infected lysogenic cells is suppressed prior to expression of the holin gene.

The putative Bam35 regulatory gene (ORF6) is similar to the E. coli LexA suppressor. We did not observe any changes in the genome region including this gene when the sequence of the clear-plaque variant was compared to the prophage genomes. Therefore, the lysogeny regulation may not occur by mechanisms involving DNA arrangements.

The present study focused on the Bam35 interactions with resistant, lysogenic, and nonlysogenic host cells. We were able to describe the PM permeability changes during viral DNA entry and when infected cells were programmed to lyse. The availability of lysogenic hosts extended our analysis to delineate the events associated with the control of lysogeny in a novel system where prophages with genome terminal proteins reside as linear plasmids within the host cell.

 
This investigation was supported by research grants 1202108 and 1202855 to D.H.B. and 1201964 to J.K.H.B. from the Academy of Finland (Finnish Center of Excellence Program [2000-2005]). A.G. was partially supported by CIMO (Center for International Mobility). R.D. is a Lithuanian State Fellowship holder.

 
* Corresponding author. Mailing address: Viikki Biocenter, 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, May 2005, p. 3521-3527, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3521-3527.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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