Structural and functional insights into the regulation of the lysis-lysogeny decision in viral communities.

Communication is vital for all organisms including microorganisms, which is clearly demonstrated by the bacterial quorum-sensing system. However, the molecular mechanisms underlying communication among viruses (phages) via the quorum-sensing-like 'arbitrium' system remain unclear. Viral or host densities are known to be related to an increased prevalence of lysogeny; however, how the switch from the lytic to the lysogenic pathway occurs is unknown. Thus, we sought to reveal mechanisms of communication among viruses and determine the lysogenic dynamics involved. Structural and functional analyses of the phage-derived SAIRGA and GMPRGA peptides and their corresponding receptors, phAimR and spAimR, indicated that SAIRGA directs the lysis-lysogeny decision of phi3T by modulating conformational changes in phAimR, whereas GMPRGA regulates the lysis-lysogeny pathway by stabilizing spAimR in the dimeric state. Although temperate viruses are thought to share a similar lytic-lysogenic cycle switch model, our study suggests the existence of alternative strain-specific mechanisms that regulate the lysis-lysogeny decision. Collectively, these findings provide insights into the molecular mechanisms underlying communication among viruses, offering theoretical applications for the treatment of infectious viral diseases.

Structural basis of the arbitrium peptide-AimR communication system in the phage lysis-lysogeny decision.

A bacteriophage can replicate and release virions from a host cell in the lytic cycle or switch to a lysogenic process in which the phage integrates itself into the host genome as a prophage. In Bacillus cells, some types of phages employ the arbitrium communication system, which contains an arbitrium hexapeptide, the cellular receptor AimR and the lysogenic negative regulator AimX. This system controls the decision between the lytic and lysogenic cycles. However, both the mechanism of molecular recognition between the arbitrium peptide and AimR and how downstream gene expression is regulated remain unknown. Here, we report crystal structures for AimR from the SPbeta phage in the apo form and the arbitrium peptide-bound form at 2.20 Å and 1.92 Å, respectively. With or without the peptide, AimR dimerizes through the C-terminal capping helix. AimR assembles a superhelical fold and accommodates the peptide encircled by its tetratricopeptide repeats, which is reminiscent of RRNPP family members from the quorum-sensing system. In the absence of the arbitrium peptide, AimR targets the upstream sequence of the aimX gene; its DNA binding activity is prevented following peptide binding. In summary, our findings provide a structural basis for peptide recognition in the phage lysis-lysogeny decision communication system.

Directional mechanical stability of Bacteriophage φ29 motor's 3WJ-pRNA: Extraordinary robustness along portal axis.

The molecular motor exploited by bacteriophage φ29 to pack DNA into its capsid is regarded as one of the most powerful mechanical devices present in viral, bacterial, and eukaryotic systems alike. Acting as a linker element, a prohead RNA (pRNA) effectively joins the connector and ATPase (adenosine triphosphatase) components of the φ29 motor. During DNA packing, this pRNA needs to withstand enormous strain along the capsid's portal axis-how this remarkable stability is achieved remains to be elucidated. We investigate the mechanical properties of the φ29 motor's three-way junction (3WJ)-pRNA using a combined steered molecular dynamics and atomic force spectroscopy approach. The 3WJ exhibits strong resistance to stretching along its coaxial helices, demonstrating its super structural robustness. This resistance disappears, however, when external forces are applied to the transverse directions. From a molecular standpoint, we demonstrate that this direction-dependent stability can be attributed to two Mg clamps that cooperate and generate mechanical resistance in the pRNA's coaxial direction. Our results suggest that the asymmetric nature of the 3WJ's mechanical stability is entwined with its biological function: Enhanced rigidity along the portal axis is likely essential to withstand the strain caused by DNA condensation, and flexibility in other directions should aid in the assembly of the pRNA and its association with other motor components.

Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor.

Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate.

Virus evolution toward limited dependence on nonessential functions of the host: the case of bacteriophage SPP1.

UNLABELLED: All viruses are obligate intracellular parasites and depend on certain host cell functions for multiplication. However, the extent of such dependence and the exact nature of the functions provided by the host cell remain poorly understood. Here, we investigated if nonessential Bacillus subtilis genes are necessary for multiplication of bacteriophage SPP1. Screening of a collection of 2,514 single-gene knockouts of nonessential B. subtilis genes yielded only a few genes necessary for efficient SPP1 propagation. Among these were genes belonging to the yuk operon, which codes for the Esat-6-like secretion system, including the SPP1 receptor protein YueB. In addition, we found that SPP1 multiplication was negatively affected by the absence of two other genes, putB and efp. The gene efp encodes elongation factor P, which enhances ribosome activity by alleviating translational stalling during the synthesis of polyproline-containing proteins. PutB is an enzyme involved in the proline degradation pathway that is required for infection in the post-exponential growth phase of B. subtilis, when the bacterium undergoes a complex genetic reprogramming. The putB knockout shortens significantly the window of opportunity for SPP1 infection during the host cell life cycle. This window is a critical parameter for competitive phage multiplication in the soil environment, where B. subtilis rarely meets conditions for exponential growth. Our results in combination with those reported for other virus-host systems suggest that bacterial viruses have evolved toward limited dependence on nonessential host functions. IMPORTANCE: A successful viral infection largely depends on the ability of the virus to hijack cellular machineries and to redirect the flow of building blocks and energy resources toward viral progeny production. However, the specific virus-host interactions underlying this fundamental transformation are poorly understood. Here, we report on the first systematic analysis of virus-host cross talk during bacteriophage infection in Gram-positive bacteria. We show that lytic bacteriophage SPP1 is remarkably independent of nonessential genes of its host, Bacillus subtilis, with only a few cellular genes being necessary for efficient phage propagation. We hypothesize that such limited dependence of the virus on its host results from a constant "evolutionary arms race" and might be much more widespread than currently thought.

Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging.

Many viruses use molecular motors that generate large forces to package DNA to near-crystalline densities inside preformed viral proheads. Besides being a key step in viral assembly, this process is of interest as a model for understanding the physics of charged polymers under tight 3D confinement. A large number of theoretical studies have modeled DNA packaging, and the nature of the molecular dynamics and the forces resisting the tight confinement is a subject of wide debate. Here, we directly measure the packaging of single DNA molecules in bacteriophage phi29 with optical tweezers. Using a new technique in which we stall the motor and restart it after increasing waiting periods, we show that the DNA undergoes nonequilibrium conformational dynamics during packaging. We show that the relaxation time of the confined DNA is >10 min, which is longer than the time to package the viral genome and 60,000 times longer than that of the unconfined DNA in solution. Thus, the confined DNA molecule becomes kinetically constrained on the timescale of packaging, exhibiting glassy dynamics, which slows the motor, causes significant heterogeneity in packaging rates of individual viruses, and explains the frequent pausing observed in DNA translocation. These results support several recent hypotheses proposed based on polymer dynamics simulations and show that packaging cannot be fully understood by quasistatic thermodynamic models.

A touch of glue to complete bacteriophage assembly: the tail-to-head joining protein (THJP) family.

Bacteriophage SPP1 is a nanomachine built to infect the bacterium Bacillus subtilis. The phage particle is composed of an icosahedric capsid, which contains the viral DNA, and a long non-contractile tail. Capsids and tails are produced in infected cells by two distinct morphogenetic pathways. Characterization of the suppressor-sensitive mutant SPP1sus82 showed that it produces DNA-filled capsids and tails but is unable to assemble complete virions. Its purified tails have a normal length but lack a narrow ring that tapers the tail end found at the tail-to-head interface. The mutant is defective in production of gp17. The gp17 ring is exposed in free tails competent for viral assembly but becomes shielded in the final virion structure. Recombinant gp17 is active in an in vitro assay to stick together capsids and tails present in extracts of SPP1sus82-infected cells, leading to formation of infectious particles. Gp17 thus plays a fundamental role in the tail-to-head joining reaction, the ultimate step of virus particle assembly. This is the conserved function of gp17 and its structurally related proteins like lambda gpU. This family of proteins can also provide fidelity to termination of the tail tube elongation reaction in a subset of phages including coliphage lambda.

Complete nucleotide sequence of Bacillus subtilis (natto) bacteriophage PM1, a phage associated with disruption of food production.

"Natto", considered a traditional food, is made by fermenting boiled soybeans with Bacillus subtilis (natto), which is a natto-producing strain related to B. subtilis. The production of natto is disrupted by phage infections of B. subtilis (natto); hence, it is necessary to control phage infections. PM1, a phage of B. subtilis (natto), was isolated during interrupted natto production in a factory. In a previous study, PM1 was classified morphologically into the family Siphoviridae, and its genome, comprising approximately 50 kbp of linear double-stranded DNA, was assumed to be circularly permuted. In the present study, the complete nucleotide sequence of the PM1 genomic DNA of 50,861 bp (41.3 %G+C) was determined, and 86 open reading frames (ORFs) were deduced. Forty-one ORFs of PM1 shared similarities with proteins deduced from the genome of phages reported so far. Twenty-three ORFs of PM1 were associated with functions related to the phage multiplication process of gene control, DNA replication/modification, DNA packaging, morphogenesis, and cell lysis. Bacillus subtilis (natto) produces a capsular polypeptide of glutamate with a γ-linkage (called poly-γ-glutamate), which appears to serve as a physical barrier to phage adsorption. One ORF of PM1 had similarity with a poly-γ-glutamate hydrolase, which is assumed to degrade the capsular barrier to allow phage progenies to infect encapsulated host cells. The genome analysis of PM1 revealed the characteristics of the phage that are consistent as Bacillus subtilis (natto)-infecting phage.

Characterization of the Holliday junction resolving enzyme encoded by the Bacillus subtilis bacteriophage SPP1.

Recombination-dependent DNA replication, which is a central component of viral replication restart, is poorly understood in Firmicutes bacteriophages. Phage SPP1 initiates unidirectional theta DNA replication from a discrete replication origin (oriL), and when replication progresses, the fork might stall by the binding of the origin binding protein G38P to the late replication origin (oriR). Replication restart is dependent on viral recombination proteins to synthesize a linear head-to-tail concatemer, which is the substrate for viral DNA packaging. To identify new functions involved in this process, uncharacterized genes from phage SPP1 were analyzed. Immediately after infection, SPP1 transcribes a number of genes involved in recombination and replication from P(E2) and P(E3) promoters. Resequencing the region corresponding to the last two hypothetical genes transcribed from the P(E2) operon (genes 44 and 45) showed that they are in fact a single gene, re-annotated here as gene 44, that encodes a single polypeptide, named gene 44 product (G44P, 27.5 kDa). G44P shares a low but significant degree of identity in its C-terminal region with virus-encoded RusA-like resolvases. The data presented here demonstrate that G44P, which is a dimer in solution, binds with high affinity but without sequence specificity to several double-stranded DNA recombination intermediates. G44P preferentially cleaves Holliday junctions, but also, with lower efficiency, replicated D-loops. It also partially complemented the loss of RecU resolvase activity in B. subtilis cells. These in vitro and in vivo data suggest a role for G44P in replication restart during the transition to concatemeric viral replication.

The opening of the SPP1 bacteriophage tail, a prevalent mechanism in Gram-positive-infecting siphophages.

The SPP1 siphophage uses its long non-contractile tail and tail tip to recognize and infect the Gram-positive bacterium Bacillus subtilis. The tail-end cap and its attached tip are the critical components for host recognition and opening of the tail tube for genome exit. In the present work, we determined the cryo-electron microscopic (cryo-EM) structure of a complex formed by the cap protein gp19.1 (Dit) and the N terminus of the downstream protein of gp19.1 in the SPP1 genome, gp21(1-552) (Tal). This complex assembles two back-to-back stacked gp19.1 ring hexamers, interacting loosely, and two gp21(1-552) trimers interacting with gp19.1 at both ends of the stack. Remarkably, one gp21(1-552) trimer displays a "closed" conformation, whereas the second is "open" delineating a central channel. The two conformational states dock nicely into the EM map of the SPP1 cap domain, respectively, before and after DNA release. Moreover, the open/closed conformations of gp19.1-gp21(1-552) are consistent with the structures of the corresponding proteins in the siphophage p2 baseplate, where the Tal protein (ORF16) attached to the ring of Dit (ORF15) was also found to adopt these two conformations. Therefore, the present contribution allowed us to revisit the SPP1 tail distal-end architectural organization. Considering the sequence conservation among Dit and the N-terminal region of Tal-like proteins in Gram-positive-infecting Siphoviridae, it also reveals the Tal opening mechanism as a hallmark of siphophages probably involved in the generation of the firing signal initiating the cascade of events that lead to phage DNA release in vivo.

The genome of Bacillus subtilis phage SP10: a comparative analysis with phage SPO1.

A nucleotide sequence of the whole genome of Bacillus subtilis phage SP10 was determined. It was composed of 143,986 bp with 236 putative open reading frames (ORFs). Sixty-five of 236 predicted ORFs showed high similarity to that of SPO1, and the genome organizations of the two phages were similar to each other. SP10 belongs to the Myoviridae family, for which the well-studied phage SPO1 is the representative phage. Hence, we compared SP10 to SPO1. The SP10 genome DNA showed different sensitivity to restriction enzymes than SPO1, due to differences in base modification. According to transcriptional analysis, the gene expression of regulatory network of SP10 was similar to SPO1. It was observed that RNA polymerase containing sigma-A was inactive in directing the host genes but active in directing the phage genes. It appeared that the association of sigma-A with the core enzyme complex of RNA polymerase was strengthened during development.

Substrate interactions and promiscuity in a viral DNA packaging motor.

The ASCE (additional strand, conserved E) superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life. A subset of these enzymes consists of multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses and tailed bacteriophages. Although their mechanism of mechanochemical conversion is beginning to be understood, little is known about how these motors engage their nucleic acid substrates. Questions remain as to whether the motors contact a single DNA element, such as a phosphate or a base, or whether contacts are distributed over several parts of the DNA. Furthermore, the role of these contacts in the mechanochemical cycle is unknown. Here we use the genome packaging motor of the Bacillus subtilis bacteriophage varphi29 (ref. 4) to address these questions. The full mechanochemical cycle of the motor, in which the ATPase is a pentameric-ring of gene product 16 (gp16), involves two phases-an ATP-loading dwell followed by a translocation burst of four 2.5-base-pair (bp) steps triggered by hydrolysis product release. By challenging the motor with a variety of modified DNA substrates, we show that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5'-3' strand in the direction of packaging. As well as providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, nonspecific contacts may reflect common translocase-substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily.

Intersubunit coordination in a homomeric ring ATPase.

Homomeric ring ATPases perform many vital and varied tasks in the cell, ranging from chromosome segregation to protein degradation. Here we report the direct observation of the intersubunit coordination and step size of such a ring ATPase, the double-stranded-DNA packaging motor in the bacteriophage phi29. Using high-resolution optical tweezers, we find that packaging occurs in increments of 10 base pairs (bp). Statistical analysis of the preceding dwell times reveals that multiple ATPs bind during each dwell, and application of high force reveals that these 10-bp increments are composed of four 2.5-bp steps. These results indicate that the hydrolysis cycles of the individual subunits are highly coordinated by means of a mechanism novel for ring ATPases. Furthermore, a step size that is a non-integer number of base pairs demands new models for motor-DNA interactions.

Origin and function of the two major tail proteins of bacteriophage SPP1.

The majority of bacteriophages have a long non-contractile tail (Siphoviridae) that serves as a conduit for viral DNA traffic from the phage capsid to the host cell at the beginning of infection. The 160-nm-long tail tube of Bacillus subtilis bacteriophage SPP1 is shown to be composed of two major tail proteins (MTPs), gp17.1 and gp17.1*, at a ratio of about 3:1. They share a common amino-terminus, but the latter species has approximately 10 kDa more than gp17.1. A CCC.UAA sequence with overlapping proline codons at the 3' end of gene 17.1 drives a programmed translational frameshift to another open reading frame. The recoding event generates gp17.1*. Phages carrying exclusively gp17.1 or gp17.1* are viable, but tails are structurally distinct. gp17.1 and the carboxyl-terminus of gp17.1* have a distinct evolutionary history correlating with different functions: the polypeptide sequence identical in the two proteins is responsible for assembly of the tail tube while the additional module of gp17.1* shields the structure exterior exposed to the environment. The carboxyl-terminal extension is an elaboration present in some tailed bacteriophages. Different extensions were found to combine in a mosaic fashion with the MTP essential module in a subset of Siphoviridae genomes.

Multifunctional roles of a bacteriophage phi 29 morphogenetic factor in assembly and infection.

Low copy number proteins within macromolecular complexes, such as viruses, can be critical to biological function while comprising a minimal mass fraction of the complex. The Bacillus subtilis double-stranded DNA bacteriophage phi 29 gene 13 product (gp13), previously undetected in the virion, was identified and localized to the distal tip of the tail knob. Western blots and immuno-electron microscopy detected a few copies of gp13 in phi 29, DNA-free particles, purified tails, and defective particles produced in suppressor-sensitive (sus) mutant sus13(330) infections. Particles assembled in the absence of intact gp13 (sus13(342) and sus13(330)) had the gross morphology of phi 29 but were not infectious. gp13 has predicted structural homology and sequence similarity to the M23 metalloprotease LytM. Poised at the tip of the phi 29 tail knob, gp13 may serve as a plug to help restrain the highly pressurized packaged genome. Also, in this position, gp13 may be the first virion protein to contact the cell wall in infection, acting as a pilot protein to depolymerize the cell wall. gp13 may facilitate juxtaposition of the tail knob onto the cytoplasmic membrane and the triggering of genome injection.

Structural and functional analysis of phi29 p16.7C dimerization mutants: identification of a novel aromatic cage dimerization motif.

Prokaryotic DNA replication is compartmentalized at the cellular membrane. The Bacillus subtilis phage varphi29-encoded membrane protein p16.7 is one of the few proteins known to be involved in the organization of prokaryotic membrane-associated DNA replication. The functional DNA binding domain of p16.7 is constituted by its C-terminal half, p16.7C, which forms high affinity dimers in solution and which can form higher order oligomers. Recently, the solution and crystal structures of p16.7C and the crystal structure of the p16.7C-DNA complex have been solved. Here, we have studied the p16.7C dimerization process and the structural and functional roles of p16.7 residues Trp-116 and Asn-120 and its last nine C-terminal amino acids, which form an extended tail. The results obtained show that transition of folded dimers into unfolded monomers occurs without stable intermediates and that both Trp-116 and the C-terminal tail are important for dimerization and functionality of p16.7C. Residue Trp-116 is involved in formation of a novel aromatic cage dimerization motif, which we call "Pro cage." Finally, whereas residue Asn-120 plays a minor role in p16.7C dimerization, we show that it is critical for both oligomerization and DNA binding, providing further evidence that DNA binding and oligomerization of p16.7C are coupled processes.

Bacillus subtilis bacteriophage SPP1-encoded gene 34.1 product is a recombination-dependent DNA replication protein.

SPP1-encoded replication and recombination proteins, involved in the early steps of the initiation of concatemeric DNA synthesis, have been analyzed. Dimeric G34.1P exonuclease degrades, with a 5' to 3' polarity and in a Mg2+-dependent reaction, preferentially linear double-stranded (ds) DNA rather than single-stranded (ss) DNA. Binding of the replisome organizer, G38P, to its cognate sites (oriDNA) halts the 5' to 3' exonucleolytic activity of G34.1P on dsDNA. The G35P recombinase increases the affinity of G34.1P for dsDNA, and stimulates G34.1P activity on dsDNA, but not on ssDNA. Then, filamented G35P promotes limited strand exchange with a homologous sequence. The ssDNA binding protein, G36P, protects ssDNA from the G34.1P exonuclease activity and stimulates G35P-catalyzed strand exchange. The data presented suggest a model for the role of G34.1P during initiation of sigma replication: G38P bound to oriDNA might halt replication fork progression, and G35P, G34.1P and G36P in concert might lead to the re-establishment of a unidirectional recombination-dependent replication that accounts for the direction of DNA packaging.

Cloning, expression, purification, and characterisation of the dUTPase encoded by the integrated Bacillus subtilis temperate bacteriophage SPbeta.

The cDNA encoding dUTPase, an enzyme catalysing the hydrolysis of dUTP to dUMP and pyrophosphate, from the integrated Bacillus subtilis temperate bacteriophage SPbeta has been cloned and over-expressed at high levels in Escherichia coli. The resulting recombinant dUTPase was purified to homogeneity in one step by phosphocellulose chromatography with a final yield of 700 mg pure crystallisable protein per litre of bacterial culture. The molecular mass of the 142 amino acid polypeptide was 16 kDa as judged by electrophoretic analysis and gel filtration chromatography revealed the enzyme to exist as a homotrimer in solution. Isoelectric focusing indicated the isoelectric point to be 7. Functionality of the purified recombinant dUTPase was proven by demonstrating catalytic activity towards the substrate dUTP. The optimal activity of SPbeta dUTPase proved to be dependent on the presence of divalent metal ions, with Mg(2+) conferring the highest activity.

The high-resolution functional map of bacteriophage SPP1 portal protein.

An essential component in the assembly of nucleocapsids of tailed bacteriophages and of herpes viruses is the portal protein that is located at the unique vertex of the icosahedral capsid through which DNA movements occur. A library of mutations in the bacteriophage SPP1 portal protein (gp6) was generated by random mutagenesis of gene 6. Screening of the library allowed identification of 67 single amino acid substitutions that impair portal protein function. Most of the mutations cluster within stretches of a few amino acids in the gp6 carboxyl-terminus. The mutations were divided into five classes according to the step of virus assembly that they impair: (1) production of stable gp6; (2) interaction of gp6 with the minor capsid protein gp7; (3) incorporation of gp6 in the procapsid structure; (4) DNA packaging; and (5) sizing of the packaged DNA molecule. Most of the mutations fell in classes 3 and 4. This is the first high-resolution functional map of a portal protein, in which its function at different steps of viral assembly can be directly correlated with specific regions of its sequence. The work provides a framework for the understanding of central processes in the assembly of viruses that use specialized portals to govern entry and exit of DNA from the viral capsid.

Characterization of poly-gamma-glutamate hydrolase encoded by a bacteriophage genome: possible role in phage infection of Bacillus subtilis encapsulated with poly-gamma-glutamate.

Some Bacillus subtilis strains, including natto (fermented soybeans) starter strains, produce a capsular polypeptide of glutamate with a gamma-linkage, called poly-gamma-glutamate (gamma-PGA). We identified and purified a monomeric 25-kDa degradation enzyme for gamma-PGA (designated gamma-PGA hydrolase, PghP) from bacteriophage PhiNIT1 in B. subtilis host cells. The monomeric PghP internally hydrolyzed gamma-PGA to oligopeptides, which were then specifically converted to tri-, tetra-, and penta-gamma-glutamates. Monoiodoacetate and EDTA both inhibited the PghP activity, but Zn(2+) or Mn(2+) ions fully restored the enzyme activity inhibited by the chelator, suggesting that a cysteine residue(s) and these metal ions participate in the catalytic mechanism of the enzyme. The corresponding pghP gene was cloned and sequenced from the phage genome. The deduced PghP sequence (208 amino acids) with a calculated M(r) of 22,939 was not significantly similar to any known enzyme. Thus, PghP is a novel gamma-glutamyl hydrolase. Whereas phage PhiNIT1 proliferated in B. subtilis cells encapsulated with gamma-PGA, phage BS5 lacking PghP did not survive well on such cells. Moreover, all nine phages that contaminated natto during fermentation produced PghP, supporting the notion that PghP is important in the infection of natto starters that produce gamma-PGA. Analogous to polysaccharide capsules, gamma-PGA appears to serve as a physical barrier to phage absorption. Phages break down the gamma-PGA barrier via PghP so that phage progenies can easily establish infection in encapsulated cells.