Evidence for a holin-like protein gene fully embedded out of frame in the endolysin gene of Staphylococcus aureus bacteriophage 187.

We have cloned, sequenced, and characterized the genes encoding the lytic system of the unique Staphylococcus aureus phage 187. The endolysin gene ply187 encodes a large cell wall-lytic enzyme (71.6 kDa). The catalytic site, responsible for the hydrolysis of staphylococcal peptidoglycan, was mapped to the N-terminal domain of the protein by the expression of defined ply187 domains. This enzymatically active N terminus showed convincing amino acid sequence homology to an N-acetylmuramoyl-L-alanine amidase, whereas the C-terminal part, whose function is unknown, revealed striking relatedness to major staphylococcal autolysins. An additional reading frame was identified entirely embedded out of frame (+1) within the 5' region of ply187 and was shown to encode a small, hydrophobic protein of holin-like function. The hol187 gene features a dual-start motif, possibly enabling the synthesis of two products of different lengths (57 and 55 amino acids, respectively). Overproduction of Hol187 in Escherichia coli resulted in growth retardation, leakiness of the cytoplasmic membrane, and loss of de novo ATP synthesis. Compared to other holins identified to date, Hol187 completely lacks the highly charged C terminus. The secondary structure of the polypeptide is predicted to consist of two small, antiparallel, hydrophobic, transmembrane helices. These are supposed to be essential for integration into the membrane, since site-specific introduction of negatively charged amino acids into the first transmembrane domain (V7D G8D) completely abolished the function of the Hol187 polypeptide. With antibodies raised against a synthetic 18-mer peptide representing a central part of the protein, it was possible to detect Hol187 in the cytoplasmic membrane of phage-infected S. aureus cells. An important indication that the protein actually functions as a holin in vivo was that the gene (but not the V7D G8D mutation) was able to complement a phage lambda Sam mutation in a nonsuppressing E. coli HB101 background. Plaque formation by lambdagt11::hol187 indicated that both phage genes have analogous functions. The data presented here indicate that a putative holin is encoded on a different reading frame within the enzymatically active domain of ply187 and that the holin is synthesized during the late stage of phage infection and found in the cytoplasmic membrane, where it causes membrane lesions which are thought to enable access of Ply187 to the peptidoglycan of phage-infected Staphylococcus cells.     

Two beginnings for a single purpose: the dual-start holins in the regulation of phage lysis

For most large phages of both Gram-positive and Gram-negative bacteria, there appears to be a single pathway for achieving disruption of the host envelope, requiring at least two phage-encoded lysis functions (a holin and an endolysin). The holin is a small membrane protein which causes a non-specific lesion in the cytoplasmic membrane, which allows the endolysin to gain access to its substrate, the peptidoglycan. The scheduling of host lysis is effected by regulatory mechanisms which govern the synthesis and activity of the holin protein accumulating in the membrane. Accordingly, aspects of expression and function of holin genes are considered here, focusing mainly on the lambdoid S genes. This group of genes, of which lambda S is the prototype, are characterized by a dual-start motif consisting of two Met start codons separated by one or two codons, at least one of which specifies Arg or Lys. Two protein products are elaborated, differing only by two or three N-terminal residues but apparently possessing opposing functions: the shorter polypeptide is the active holin, or lysis-effector, whereas the longer polypeptide apparently acts as an inhibitor of holin function. Models will be considered which may account for the ability of the holin to form a 'hole' in the cytoplasmic membrane at a programmed time, as well as for the inhibitory properties of the longer product. Finally, we discuss recent results suggesting that the dual-start motif can be viewed as a level of regulation superimposed on a timing function intrinsic to the canonical holin structure.     

Functional Analysis of a Class I Holin,P2 Y

Y is the putative holin gene of the paradigm coliphage P2 and encodes a 93-amino-acid protein. Y is predicted to be an integral membrane protein that adopts an N-out C-in membrane topology with 3 transmembrane domains (TMDs) and a highly charged C-terminal cytoplasmic tail. The same features are observed in the canonical class I lambda holin, the S105 protein of phage lambda, which controls lysis by forming holes in the plasma membrane at a programmed time. S105 has been the subject of intensive genetic, cellular, and biochemical analyses. Although Y is not related to S105 in its primary structure, its characterization might prove useful in discerning the essential traits for holin function. Here, we used physiological and genetic approaches to show that Y exhibits the essential holin functional criteria, namely, allele-specific delayed-onset lethality and sensitivity to the energization of the membrane. Taken together, these results suggest that class I holins share a set of unusual features that are needed for their remarkable ability to program the end of the phage infection cycle with precise timing. However, Y holin function requires the integrity of its short cytoplasmic C-terminal domain, unlike for S105. Finally, instead of encoding a second translational product of Y as an antiholin, as shown for lambda S107, the P2 lysis cassette encodes another predicted membrane protein, LysA, which is shown here to have a Y-specific antiholin character.     

Biochemical and genetic evidence for three transmembrane domains in the class I holin, lambda S

lambda S, the prototype class I holin gene, encodes three potential transmembrane domains in its 107 codons, whereas 21 S, the class II prototype spans only 71 codons and encodes two transmembrane domains. Many holin genes, including lambda S and 21 S, have the "dual-start" regulatory motif at the N terminus, suggesting that class I and II holins have the same topology. The primary structure of 21 S strongly suggests a bitopic "helical-hairpin" topology, with N and C termini on the cytoplasmic side of the membrane. However, lambda S chimeras with an N-terminal signal sequence show Lep-dependent function, indicating that the N-terminal domain of S requires export. Here the signal sequence chimera is shown to be sensitive to the missense change A52V, which blocks normal S function. Moreover, cysteine-modification studies in isolated membranes using a collection of S variants with single-cysteine substitutions show that the positions in the core of the 3 putative transmembrane domains of lambda S are protected. Also, S proteins with single-cysteine substitutions in the predicted cytoplasmic and periplasmic loops are more efficiently labeled in inverted membrane vesicles and whole cells, respectively. These data constitute direct evidence that the holin S(lambda) has three transmembrane domains and indicate that class I and class II holins have different topologies, despite regulatory and functional homology.     

Sizing the holin lesion with an endolysin-beta-galactosidase fusion

Double-stranded DNA phages require two proteins for efficient host lysis: the endolysin, a muralytic enzyme, and the holin, a small membrane protein. In an event that defines the end of the vegetative cycle, the lambda holin S acts suddenly to permeabilize the membrane. This permeabilization enables the R endolysin to attack the cell wall, after which cell lysis occurs within seconds. A C-terminal fusion of the R endolysin with full-length beta-galactosidase (beta-Gal) was tested for lytic competence in the context of the late-gene expression system of an induced lambda lysogen. Under these conditions, the hybrid R-beta-Gal product, an active tetrameric beta-Gal greater than 480 kDa in mass, was fully functional in lysis mediated by the S holin. Western blot analysis demonstrated that the lytic competence was not due to the proteolytic release of the endolysin domain of the R-beta-Gal fusion protein. The ability of this massive complex to be released by the S holin suggests that S causes a generalized membrane disruption rather than a regular oligomeric membrane pore. Similar results were obtained with an early lysis variant of the S holin and also in parallel experiments with the T4 holin, T, in an identical lambda context. However, premature holin lesions triggered by depolarization of the membrane were nonpermissive for the hybrid endolysin, indicating that these premature lesions constituted less-profound damage to the membrane. Finally, a truncated T holin functional in lysis with the endolysin is completely incompetent for lysis with the hybrid endolysin. A model for the formation of the membrane lesion within homo-oligomeric rafts of holin proteins is discussed.     

Genetic and biochemical analysis of dimer and oligomer interactions of the lambda S holin

Bacteriophage lambda uses a holin-endolysin system for host cell lysis. R, the endolysin, has muralytic activity. S, the holin, is a small membrane protein that permeabilizes the inner membrane at a precisely scheduled time after infection and allows the endolysin access to its substrate, resulting in host cell lysis. lambda S has a single cysteine at position 51 that can be replaced by a serine without loss of the holin function. A collection of 27 single-cysteine products of alleles created from lambda S(C51S) were tested for holin function. Most of the single-cysteine variants retained the ability to support lysis. Mutations with the most defective phenotype clustered in the first two hydrophobic transmembrane domains. Several lines of evidence indicate that S forms an oligomeric structure in the inner membrane. Here we show that oligomerization does not depend on disulfide bridge formation, since the cysteineless S(C51S) (i) is functional as a holin and (ii) shows the same oligomerization pattern as the parental S protein. In contrast, the lysis-defective S(A52V) mutant dimerizes but does not form cross-linkable oligomers. Again, dimerization does not depend on the natural cysteine, since the cysteineless lysis-defective S(A52V/C51S) is found in dimers after treatment of the membrane with a cross-linking agent. Furthermore, under oxidative conditions, dimerization via the natural cysteine is very efficient for S(A52V). Both S(A52V) (dominant negative) and S(A48V) (antidominant) interact with the parental S protein, as judged by oxidative disulfide bridge formation. Thus, productive and unproductive heterodimer formation between the parental protein and the mutants S(A52V) and S(A48V), respectively, may account for the dominant and antidominant lysis phenotypes. Examination of oxidative dimer formation between S variants with single cysteines in the hydrophobic core of the second membrane-spanning domain revealed that positions 48 and 51 are on a dimer interface. These results are discussed in terms of a three-step model leading to S-dependent hole formation in the inner membrane.     

The murein hydrolase of the bacteriophage phi3626 dual lysis system is active against all tested Clostridium perfringens strains

Clostridium perfringens commonly occurs in food and feed, can produce an enterotoxin frequently implicated in food-borne disease, and has a substantial negative impact on the poultry industry. As a step towards new approaches for control of this organism, we investigated the cell wall lysis system of C. perfringens bacteriophage phi3626, whose dual lysis gene cassette consists of a holin gene and an endolysin gene. Hol3626 has two membrane-spanning domains (MSDs) and is a group II holin. A positively charged beta turn between the two MSDs suggests that both the amino terminus and the carboxy terminus of Hol3626 might be located outside the cell membrane, a very unusual holin topology. Holin function was experimentally demonstrated by using the ability of the holin to complement a deletion of the heterologous phage lambda S holin in lambdadeltaSthf. The endolysin gene ply3626 was cloned in Escherichia coli. However, protein synthesis occurred only when bacteria were supplemented with rare tRNA(Arg) and tRNA(Ile) genes. Formation of inclusion bodies could be avoided by drastically lowering the expression level. Amino-terminal modification by a six-histidine tag did not affect enzyme activity and enabled purification by metal chelate affinity chromatography. Ply3626 has an N-terminal amidase domain and a unique C-terminal portion, which might be responsible for the specific lytic range of the enzyme. All 48 tested strains of C. perfringens were sensitive to the murein hydrolase, whereas other clostridia and bacteria belonging to other genera were generally not affected. This highly specific activity towards C. perfringens might be useful for novel biocontrol measures in food, feed, and complex microbial communities.     

Bacteriophage lysis: mechanism and regulation.

Bacteriophage lysis involves at least two fundamentally different strategies. Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin in this review. The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer. This is necessary because phage-encoded lysins never have secretory signal sequences and are thus incapable of unassisted escape from the cytoplasm. The holins, whose prototype is the lambda S protein, share a common organization in terms of the arrangement of charged and hydrophobic residues, and they may all contain at least two transmembrane helical domains. The available evidence suggests that holins oligomerize to form nonspecific holes and that this hole-forming step is the regulated step in phage lysis. The correct scheduling of the lysis event is as much an essential feature of holin function as is the hole formation itself. In the second strategy of lysis, used by the small single-stranded DNA phage phi X174 and the single-stranded RNA phage MS2, no murein hydrolase activity is synthesized. Instead, there is a single species of small membrane protein, unlike the holins in primary structure, which somehow causes disruption of the envelope. These lysis proteins function by activation of cellular autolysins. A host locus is required for the lytic function of the phi X174 lysis gene E.     

Genetic Dissection of T4 Lysis

t is the holin gene for coliphage T4, encoding a 218-amino-acid (aa) protein essential for the inner membrane hole formation that initiates lysis and terminates the phage infection cycle. T is predicted to be an integral membrane protein that adopts an Nⁱⁿ-C^out topology with a single transmembrane domain (TMD). This holin topology is different from those of the well-studied holins S105 (3 TMDs; N^out-Cⁱⁿ) of the coliphage lambda and S68 (2 TMDs; Nⁱⁿ-Cⁱⁿ) of the lambdoid phage 21. Here, we used random mutagenesis to construct a library of lysis-defective alleles of t to discern residues and domains important for holin function and for the inhibition of lysis by the T4 antiholin, RI. The results show that mutations in all 3 topological domains (N-terminal cytoplasmic, TMD, and C-terminal periplasmic) can abrogate holin function. Additionally, several lysis-defective alleles in the C-terminal domain are no longer competent in binding RI. Taken together, these results shed light on the roles of the previously uncharacterized N-terminal and C-terminal domains in lysis and its real-time regulation.     

Probing the Structure of the S105 Hole

For most phages, holins control the timing of host lysis. During the morphogenesis period of the infection cycle, canonical holins accumulate harmlessly in the cytoplasmic membrane until they suddenly trigger to form lethal lesions called holes. The holes can be visualized by cryo-electron microscopy and tomography as micrometer-scale interruptions in the membrane. To explore the fine structure of the holes formed by the lambda holin, S105, a cysteine-scanning accessibility study was performed. A collection of S105 alleles encoding holins with a single Cys residue in different positions was developed and characterized for lytic function. Based on the ability of 4-acetamido-4′-((iodoacetyl) amino) stilbene-2,2′-disulfonic acid, disodium salt (IASD), to modify these Cys residues, one face of transmembrane domain 1 (TMD1) and TMD3 was judged to face the lumen of the S105 hole. In both cases, the lumen-accessible face was found to correspond to the more hydrophilic face of the two TMDs. Judging by the efficiency of IASD modification, it was concluded that the bulk of the S105 protein molecules were involved in facing the lumen. These results are consistent with a model in which the perimeters of the S105 holes are lined by the holin molecules present at the time of lysis. Moreover, the findings that TMD1 and TMD3 face the lumen, coupled with previous results showing TMD2-TMD2 contacts in the S105 dimer, support a model in which membrane depolarization drives the transition of S105 from homotypic to heterotypic oligomeric interactions.     

Dimerization between the holin and holin inhibitor of phage lambda

Holins are integral membrane proteins that control the access of phage-encoded muralytic enzymes, or endolysins, to the cell wall by the sudden formation of an uncharacterized homo-oligomeric lesion, or hole, in the membrane, at a precisely defined time. The timing of lambda-infected cell lysis depends solely on the 107 codon S gene, which encodes two proteins, S105 and S107, which are the holin and holin inhibitor, respectively. Here we report the results of biochemical and genetic studies on the interaction between the holin and the holin inhibitor. A unique cysteine at position 51, in the middle of the second transmembrane domain, is shown to cause the formation of disulfide-linked dimers during detergent membrane extraction. Forced oxidation of membranes containing S molecules also results in the formation of covalently linked dimers. This technique is used to demonstrate efficient dimeric interactions between S105 and S107. These results, coupled with the previous finding that the timing of lysis depends on the excess of the amount of S105 over S107, suggest a model in which the inhibitor functions by titrating out the effector in a stoichiometric fashion. This provides a basis for understanding two evolutionary advantages provided by the inhibitor system, in which the production of the inhibitor not only causes a delay in the timing of lysis, allowing the assembly of more virions, but also increases effective hole formation after triggering.     

Functional analysis of the phage T4 holin in a λ context

Phage lambda hybrids were constructed by inserting the t gene of phage T4 in place of the lambda holin gene, S. Induction of the hybrid phage resulted in lysis that was just as abrupt as, but occurred much earlier in the vegetative cycle than, that obtained with lambda, indicating that t is indeed a holin gene. Moreover, it was possible to impose lysis inhibition (LIN) on induction of the hybrid phage, but not of the parental lambda phage, by superinfection with LIN-competent T4. The imposition of the LIN state was found to depend on the allelic state of the rI and t genes of the superinfecting T4 phage, indicating that the LIN-sensitive state of the T holin is transient. Finally, induction of lysogens carrying both holin genes was shown to result in earlier triggering of lysis than with either holin gene alone. This result suggests that the two very dissimilar holins contribute additively to the physiology of the timing mechanism, or, less likely, that they interact to form one mass-action pool. In either case, these results imply a common pathway for holin timing and function.     

Characterization of the dual start motif of a class II holin gene

Holins are small membrane proteins that, at a genetically programmed time in a bacteriophage infective cycle, allow bacteriolytic enzymes, or endolysins, to escape to the periplasm and to attack the cell wall. Most holins fall into two sequence classes, I and II, based on the number of potential transmembrane domains (three for class I and two for class II). The prototype class I holin gene, S  ^λ, has a dual start motif and encodes not only the effector holin, S^λ105, but also an inhibitor, S^λ107, with a Met–Lys … extension at the terminus. The prototype class II holin gene of phage 21, S  ²¹, begins with the motif Met–Lys–Ser–Met … , and a potential RNA secondary structure overlaps the Shine–Dalgarno sequence. Here, we demonstrate that (i) two protein products are elaborated from S  ²¹, S²¹71 and S²¹68; (ii) the shorter product is required for lysis; (iii) the longer product, S²¹71, inhibits S  ²¹ function; and (iv) the Lys-2 residue is important for the inhibitor function. Moreover, the RNA stem–loop structure is involved in the downregulation of S²¹71 synthesis. However, our results suggest that, in S  ²¹, different segments of the single consensus Shine–Dalgarno sequence serve the two translational starts. These results show that the dual start motifs of class II holin genes are functionally homologous to those of class I holin genes.     

Functional Analysis of the Two-Gene Lysis System of the Pneumococcal Phage Cp-1 in Homologous and Heterologous Host Cells

The two lysis genes cph1 and cpl1 of the Streptococcus pneumoniae bacteriophage Cp-1 coding for holin and lysozyme, respectively, have been cloned and expressed in Escherichia coli. Synthesis of the Cph1 holin resulted in bacterial cell death but not lysis. The cph1 gene was able to complement a lambda Sam mutation in the nonsuppressing E. coli HB101 strain to produce phage progeny, suggesting that the holins encoded by both phage genes have analogous functions and that the pneumococcal holin induces a nonspecific lesion in the cytoplasmic membrane. Concomitant expression of both holin and lysin of Cp-1 in E. coli resulted in cell lysis, apparently due to the ability of the Cpl1 lysozyme to hydrolyze the peptidoglycan layer of this bacterium. The functional analysis of the cph1 and cpl1 genes cloned in a pneumococcal mutant with a complete deletion of the lytA gene, which codes for the S. pneumoniae main autolysin, provided the first direct evidence that, in this gram-positive-bacterium system, the Cpl1 endolysin is released to its murein substrate through the activity of the Cph1 holin. Demonstration of holin function was achieved by proving the release of pneumolysin to the periplasmic fraction, which strongly suggested that the holin produces a lesion in the pneumococcal membrane.     

The Lysis System of the <Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> Phage μ1/6

Previously, two genes, designated as lyt and hol, were identified in the lysis module of phage μ1/6. They were cloned and expressed in Escherichia coli. An additional candidate holin gene, hol2, was found downstream from the hol gene based on one predicted transmembrane domain and a highly charged C-terminal sequence of the encoded protein. Expression of hol or hol2 in E. coli was shown to cause cell death. The concomitant expression of λ endolysin (R) and μ1/6 holin resulted in cell lysis. Similarly, the coexpression of the endolysin and holin of phage μ1/6 led to lysis, apparently due to the ability of μ1/6 endolysin to hydrolyze the peptidoglycan layer of this bacterium. In contrast, the simultaneous expression of μ1/6 hol2 and the endolysin gene (λR or μ1/6 lyt) did not cause detectable lysis of the host cells. Demonstration of the holin function in streptomycetes was achieved by providing for the release of μ1/6 endolysin to the periplasm and subsequent cleavage of the peptidoglycan, which strongly suggested that the holin produces lesions in the streptomycete membrane.     

Identification and mutational analysis of bacteriophage PRD1 holin protein P35

Holin proteins are phage-induced integral membrane proteins which regulate the access of lytic enzymes to host cell peptidoglycan at the time of release of progeny viruses by host cell lysis. We describe the identification of the membrane-containing phage PRD1 holin gene (gene XXXV). The PRD1 holin protein (P35, 12.8 kDa) acts similarly to its functional counterpart from phage lambda (gene S), and the defect in PRD1 gene XXXV can be corrected by the presence of gene S of lambda. Several nonsense, missense, and insertion mutations in PRD1 gene XXXV were analyzed. These studies support the overall conclusion that the charged amino acids at the protein C terminus are involved in the timing of host cell lysis.     

The hydrophilic C-terminal part of the lambda S holin is non-essential for intermolecular interactions

Available evidence indicates that oligomerization of the bacteriophage lambda S holin leads to a non-specific lesion in the cytoplasmic membrane which permits transit of the phage encoded transglycosylase to the periplasm. In an attempt to locate an intermolecular interaction domain in S a chimeric protein comprising the N-terminal 32 aa of phage PhiX174 lysis protein E and the last 75 aa of lambda S has been constructed. We report that the EΦS fusion protein is stable, membrane bound, and inhibits S-mediated lysis in trans. C-terminal truncations of the EΦS fusion protein indicated that the hydrophilic C-terminal end of S (i.e. the last 15 aa) is non-essential for oligomerization.     

An ancient player unmasked: T4 rI encodes a t-specific antiholin

Phage T4 effects lysis by its holin T and its endolysin E. Lysis is inhibited (LIN) if the infected cell is subjected to secondary infections by T4 phage particles. The T4 rI gene is required for LIN in all hosts tested. Here, we show that a cloned rI gene can impose a T-specific LIN on T-mediated lysis in the context of the phage lambda infective cycle, in the absence of other T4 genes and without secondary infection by T4. Moreover, it is shown that the T holin accumulates in the membrane during LIN, forming SDS-resistant oligomers. We show by cross-linking experiments that a T-RI heterodimer is formed during LIN, demonstrating that RI belongs to the functional class of antiholins, such as the S107 protein of lambda, which heterodimerizes with its cognate holin, S105. Finally, we show that the addition of Ni(2+) ions to the medium can block lysis by a T protein hexahistidine-tagged at its C-terminus, suggesting that liganding of the periplasmic domain is sufficient to impose lysis inhibition. The results are discussed in terms of a model in which the LIN-inducing signal of the secondary infecting phage influences a conformational equilibrium assumed by RI in the periplasm.