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.     

Purification and Biochemical Characterization of the Lambda Holin

Holins are small phage-encoded cytoplasmic membrane proteins, remarkable for their ability to make membranes permeable in a temporally regulated manner. The purification of S105, the λ holin, and one of the two products of gene S is described. Because the wild-type S105 holin could be only partially purified from membrane extracts by ion-exchange chromatography, an oligohistidine tag was added internally to the S105 sequence for use in immobilized metal affinity chromatography. An acceptable site for the tag was found between residues 94 and 95 in the highly charged C-terminal domain of S. This allele, designated S105H94, had normal lysis timing under physiological expression conditions. The S105H94 protein was overproduced, purified, and characterized by circular dichroism spectroscopy, which revealed approximately 40% alpha-helix conformation, consistent with the presence of two transmembrane helices. The purified protein was then used to achieve release of fluorescent dye loaded in liposomes in vitro, whereas protein from an isogenic construct carrying an S mutation known to abolish hole formation was inactive in this assay. These results suggest that S is a bitopic membrane protein capable of forming aqueous holes in bilayers.     

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.     

Redox Regulation of Large Conductance Ca2+-activated K+ Channels in Smooth Muscle Cells

The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca²⁺-activated K⁺ (maxi-K) channels were examined in excised patches from tracheal myocytes. Channel activity was modified by sulfhydryl redox agents applied to the cytosolic surface, but not the extracellular surface, of membrane patches. Sulfhydryl reducing agents dithiothreitol, β-mercaptoethanol, and GSH augmented, whereas sulfhydryl oxidizing agents diamide, thimerosal, and 2,2′-dithiodipyridine inhibited, channel activity in a concentration-dependent manner. Channel stimulation by reduction and inhibition by oxidation persisted following washout of the compounds, but the effects of reduction were reversed by subsequent oxidation, and vice versa. The thiol-specific reagents N-ethylmaleimide and (2-aminoethyl)methanethiosulfonate inhibited channel activity and prevented the effect of subsequent sulfhydryl oxidation. Measurements of macroscopic currents in inside-out patches indicate that reduction only shifted the voltage/nP_o relationship without an effect on the maximum conductance of the patch, suggesting that the increase in nP_o following reduction did not result from recruitment of more functional channels but rather from changes of channel gating. We conclude that redox modulation of cysteine thiol groups, which probably involves thiol/disulfide exchange, alters maxi-K channel gating, and that this modulation likely affects channel activity under physiological conditions.     

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.     

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.     

Functional assembly of the λ S holin requires periplasmic localization of its N-terminus

Bacteriophage-λ-induced host-cell lysis requires two phage-encoded proteins, the S holin and the R transglycosylase. At a specific time during infection, the holin forms a lesion in the cytoplasmic membrane that permits access of the R protein to its substrate, the peptidoglycan. The λS gene represents the prototype of holin genes with a dual-start motif; they encode two proteins, a lysis effector and a lysis inhibitor. Although the two S proteins differ only by two amino acids (Met-1 and Lys-2) at the N-terminus, the longer product (S107) acts as an inhibitor of the lysis effector (S105). The functional difference between the proteins has been previously ascribed to the Lys-2 residue in S107. It was therefore of interest to determine the subcellular localization of the N-terminus of either S protein. To study the membrane topology of the S proteins, we used the topology probe TEM β-lactamase and an N-terminal tag derived from the Pseudomonas aeruginosa phage Pf3 coat protein. We show that both S proteins have a type III (N_out/C_in) topology. The results provide insight into the regulatory mechanism imposed by the dual-start motif and will be discussed in terms of a model for temporal regulation of the S-dependent “hole” in the membrane. Received: 28 January 1999 / Accepted: 23 April 1999     

Solubilization and delivery by GroEL of megadalton complexes of the lambda holin

GroEL can solubilize membrane proteins by binding them in its hydrophobic cavity when detergent is removed by dialysis. The best-studied example is bacteriorhodopsin, which can bind in the GroEL chaperonin at two molecules per tetradecamer. Applying this approach to the holin and antiholin proteins of phage lambda, we find that both proteins are solubilized by GroEL, in an ATP-sensitive mode, but to vastly different extents. The antiholin product, S107, saturates the chaperonin at six molecules per tetradecameric complex, whereas the holin, S105, which is missing the two N-terminal residues of S107, forms a hyper-solubilization complex with up to 350 holin molecules per GroEL, or approximately 4 MDa of protein per 0.8 MDa tetradecamer. Gel filtration chromatography and immunoprecipitation experiments confirmed the existence of complexes of the predicted masses for both S105 and S107 solubilization. For S105, negatively stained electron microscopic images show structures consistent with protein shells of the holin assembled around the chaperonin tetradecamer. Importantly, S105 can be delivered rapidly and efficiently to artificial liposomes from these complexes. In these delivery experiments, the holin exhibits efficient membrane-permeabilizing activity. The S107 antiholin can block formation of the hypersolubilization complexes, suggesting that their formation is related to an oligomerization step intrinsic to holin function.     

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.     

Extensive biodegradation of highly chlorinated biphenyl and Aroclor 1242 by Pseudomonas aeruginosa TMU56 isolated from contaminated soils

A bacterial strain, designated TMU56, was isolated from soil that had been contaminated with electrical transformer fluid (Askarel) for over 35 years. The isolate was identified as Pseudomonas aeruginosa using its 16S rDNA sequence. This strain was found to grow on monochlorobiphenyls (CBs), including 2-chlorobenzoic acid and 4-chlorobenzoic acid. It was also found to grow on 2,4-, 2,5-, 2,2′-, and 4,4′-diCB, as well as on a wide range of other xenobiotic compounds. This is the first reported representative of the genus Pseudomonas that is capable of growing on 2,4,4′-triCB, 2,2′,5,5′-tetraCB and 2,2′,4,4′,5,5′-hexaCB as sole carbon sources. Washed benzoate-grown cells were able to degrade 89% and 56% of 2,4-diCB and 2,2′,4,4′,5,5′-hexaCB, respectively. Gas chromatography analysis of individual congeners in Aroclor 1242 (200 ppm) following a 4-day incubation showed 73.3% degradation of PCBs without the need for biphenyl as an inducer. The strain exhibited no noticeable specificity for the percentage of congener transformation or degree of chlorination.     

λ噬菌体穿孔素(holin) 蛋白触发裂菌的分子机制

穿孔素-裂解酶二元裂解系统是双链DNA噬菌体普遍采用的裂菌模式,以λ噬菌体为例,系统地揭示了噬菌体穿孔素的结构与功能。λ噬菌体的S基因的特征是呈双起始基序(dual-start motif),编码穿孔素(holin)S105和抗穿孔素(antiholin)S107,通过二者不同水平的表达及相互作用,触发裂菌过程。作者综述了λ噬菌体穿孔素的膜拓扑结构和成孔机制的最新研究进展,并展望了穿孔素的研究热点和应用前景。     

The holin of bacteriophage lambda forms rings with large diameter

Holins control the length of the infection cycle of tailed phages (the Caudovirales) by oligomerizing to form lethal holes in the cytoplasmic membrane at a time dictated by their primary structure. Nothing is currently known about the physical basis of their oligomerization or the structure of the oligomers formed by any known holin. Here we use electron microscopy and single-particle analysis to characterize structures formed by the bacteriophage λ holin (S105) in vitro . In non-ionic or mild zwitterionic detergents, purified S105, but not the lysis-defective variant S105_A52V, forms rings of at least two size classes, the most common having inner and outer diameters of 8.5 and 23 nm respectively, and containing approximately 72 S105 monomers. The height of these rings, 4 nm, closely matches the thickness of the lipid bilayer. The central channel is of unprecedented size for channels formed by integral membrane proteins, consistent with the non-specific nature of holin-mediated membrane permeabilization. S105 present in detergent-solubilized rings and in inverted membrane vesicles showed similar sensitivities to proteolysis and cysteine-specific modification, suggesting that the rings are representative of the lethal holes formed by S105 to terminate the infection cycle and initiate lysis.     

The N-Terminal Transmembrane Domain of λ S Is Required for Holin but Not Antiholin Function

The λ S gene encodes a holin, S105, and an antiholin, S107, which differs by its Met-Lys N-terminal extension. The model for the lysis-defective character of S107 stipulates that the additional N-terminal basic residue keeps S107 from assuming the topology of S105, which is N-out, C-in, with three transmembrane domains (TMDs). Here we show that the N terminus of S105 retains its fMet residue but that the N terminus of S107 is fully deformylated. This supports the model that in S105, TMD1 inserts into the membrane very rapidly but that in S107, it is retained in the cytoplasm. Further, it reveals that, compared to S105, S107 has two extra positively charged moieties, Lys2 and the free N-terminal amino group, to hinder its penetration into an energized membrane. Moreover, an allele, S105_ΔTMD1, with TMD1 deleted, was found to be defective in lysis, insensitive to membrane depolarization, and dominant to the wild-type allele, indicating that the lysis-defective, antiholin character of S107 is due to the absence of TMD1 from the bilayer rather than to its ectopic localization at the inner face of the cytoplasmic membrane. Finally, the antiholin function of the deletion protein was compromised by the substitution of early-lysis missense mutations in either the deletion protein or parental S105 but restored when both S105_ΔTMD1 and holin carried the substitution.In general, holins control the length of the infection cycle of double-stranded DNA phages (37). During late gene expression, the holin protein accumulates harmlessly in the bilayer until suddenly and spontaneously triggering the formation of holes in the membrane at an allele-specific time (13, 15). Holin genes are extremely diverse, but most can be grouped into two main classes based on the number of predicted transmembrane domains (TMDs): class I, with three TMDs and a predicted N-out, C-in topology, and class II, with two TMDs and a predicted N-in, C-in topology (38). Holin genes and function are subject to several levels of regulation, among which a particularly striking feature is the common occurrence of two potential translational starts, or dual-start motifs (5, 37), separated by only a few codons. Dual-start motifs are found in many holins of both of the two major classes; in nearly every case, the two starts are separated by at least one basic residue. The first dual-start motif to be characterized was that of λ S, the prototype class I holin gene (Fig. 1A and B). Translation initiation events occur at codons 1 and 3, giving rise to two products, S107 and S105, each named because of the length of its amino acid sequence; in the wild-type (wt) allele, two RNA structures define the ratio of initiations at the two start codons, resulting in an S105/S107 ratio of ∼2:1.Open in a separate windowFIG. 1.Gene, topology, and sequence of λ S. (A, top) The λ lysis cassette, including genes S, R, Rz, and Rz1, is shown, along with its promoter p_R′, and Q, encoding the late gene activator. The 5′ end of the class I holin gene S has two start codons, Met1, the start for S107, and Met3, the start for S105, and two RNA structures that regulate initiations at these codons. The S105 and S107 alleles have Leu (CUG) codons in place of the Met3 and Met1 codons, respectively. (B) Primary structure of S proteins. Missense changes relevant to the text are shown. Starts for S107 and S105 are indicated by asterisks. The three TMDs are boxed (13), and the extent of the ΔTMD1 deletion is indicated. (C) Model for the membrane topology of S105, S107, and S105_ΔTMD1. Topology and boundary residues for TMD1, -2, and -3 are based on Graschopf and Blasi (11) and Gründling et al. (13), respectively.Although they differ only by the Met-Lys N-terminal extension of S107, the two proteins have opposing functions; S105 is the holin and S107 the antiholin. The antiholin function is reflected by four principal features: first, when the Met3 start is inactivated, the mutant allele, designated S107 (Fig. ​(Fig.1A),1A), is lysis defective (26); second, the S107 protein binds and inhibits S105 specifically (3, 16); third, when S107 is produced in stoichiometric excess over S105, lysis is blocked for several times the length of the normal infection cycle (3, 4, 7, 16); and fourth, S107 antiholin function, i.e., inhibition of S105 hole formation, can be instantly subverted by collapsing the proton motive force, most easily done by addition of energy poisons to the medium (3). The predicted N-out, C-in topology and the requirement for the energized membrane led to a model in which S107 is initially inserted in the membrane with only two TMDs, with TMD1 being blocked from insertion by the presence of the positively charged residue, Lys2, whereas S105 has three TMDs (Fig. ​(Fig.1C)1C) (39). From this perspective, S105-S107 complexes, which are approximately twice as numerous as the S105 homodimers, are defective in triggering hole formation. An appealing feature of this model is that when an S105-mediated hole formation event does occur in a cell, the resultant collapse of the membrane potential allows insertion of TMD1 of S107 into the membrane, instantly tripling the amount of active holin by making the previously inactive pool of S105-S107 complexes functional (38).Some genetic and physiological evidence for the topology of the λ S proteins has been obtained using gene fusions. First, a fusion of the S gene at codon 105 with lacZ generates a functional, membrane-inserted β-galactosidase chimera, indicating, as expected, the cytoplasmic disposition of the highly charged C terminus of the S protein (40). Second, Graschopf and Bläsi (12) demonstrated that S-mediated hole formation could be obtained with constructs where a secretory signal sequence was fused to the N termini of both S105 and S107. Lysis required the cleavage of the signal sequence by leader peptidase, and export of the signal-S107 form was slower than for the signal-S105 form. However, evidence for the topology of native forms of S has not been available. Moreover, no basis for the inhibitory character of S107 has been established. In the simplest view, the antiholin function could be due to the absence of TMD1 from the bilayer or the ectopic localization of TMD1 in the cytoplasm, or both. Here, we report studies directed at dissecting the precise role of topology in S107 function and correlating antiholin activity with its ability to heterodimerize with S105. The results are discussed in terms of a general model for the formation of the holin lesion and the role of dynamic membrane topology in its temporal regulation.     

Effects of 4-acetamido-4′-isothiocyano-2,2′-disulfonic stilbene on ion transport in turtle bladders

The disulfonic stilbene (4-acetamido-4′-isothiocyano-2,2′-disulfonic stilbene) is found to be more potent than acetazolamide as an anion transport inhibitor in the turtle bladder, but less potent than acetazolamide as a carbonic anhydrase inhibitor. The anion-dependent (HCO₃^-−, Cl⁻) moeity of the short-circuiting current is eliminated by 4-acetamido-4′-isothiocyano-2,2′-disulfonic stibene, but only after its addition to the serosal bathing fluid. Whereas 4-acetmido-4′-isothiocyano-2,2′-disulfonic stilbene has no effect om Na⁺transport across the bladder, it is more potent than ouabain as an inhibitor of microsomal (Na⁺+K⁺)-ATPase of both turtle bladder and eel electric organ.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca/Mg)-ATPase, and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

Relation between red cell membrane (Na + K)-ATPase and band 3 protein

This study is designed to examine the participation of the major red cell membrane protein, band 3 protein, in the chain which transmits information from the cardiac glycoside site on the external face of the cell (Na⁺ + K⁺)-ATPase to the megadalton glycolytic enzyme complex within the cell. The experiments show that the anion transport inhibitor, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, affects the resonance of 2,3-diphosphoglycerate, as does the cardiac glycoside cation transport inhibitor, ouabain. Resonance shifts induced by the cardiac glycoside alone are modulated by addition of the anion transport inhibitor which indicates that there is coupling in the red cell between the (Na⁺ + K⁺)-ATPase and band 3 protein. Band 3 protein was separated from the membrane and partially purified following the technique of Yu and Steck ((1975) J. Biol. Chem. 250, 9170–9175). When glyceraldehyde-3-phosphate dehydrogenase was added to the separated band 3 protein preparation, addition of cardiac glycosides caused shifts in the ³¹P resonance of glyceraldehyde 3-phosphate. These experiments indicate that there is coupling between the (Na⁺ + K⁺)-ATPase and band 3 protein in the separated preparation and suggest that the anion and cation transport systems may be closely related spatially and functionally in the intact red cell.     

P2X<Subscript>7</Subscript> Receptor Stimulation of Membrane Internalization in a Thyrocyte Cell Line

Using fluorescent membrane markers, we have previously shown that extracellular ATP stimulates both exocytosis and membrane internalization in the Fisher rat thyroid cell line FRTL. In this study, we examine the actions of ATP using whole-cell recording conditions that favor stimulation of membrane internalization. ATP stimulation of the P2X₇ receptor activated a reversible, Ca²⁺-permeable, cation conductance that slowly increased in size without changes in ion selectivity. ATP also induced a delayed irreversible decrease in cell capacitance (C_m) that was equivalent to an 8% decrease in membrane surface area. Addition of guanosine 5′-0-2-thiodiphosphate to the pipette solution inhibited the ATP-induced decrease in C_m without affecting channel activation. The effects of ATP on membrane conductance were mimicked by 2′,3′-O-(4-benzoylbenzoyl)-ATP, but not by UTP, adenosine, or 2-methylthio-ATP, and were inhibited by pyridoxal phosphate-6-azophenyl-2′4′-disulfonic acid, adenosine 5′-triphosphate-2′3′-dialdehyde, and Cu²⁺. The capacitance decrease persisted in Na⁺-, Ca²⁺- and Cl⁻-free external saline or with Ca²⁺-free pipette solution. It is concluded that ATP activation of the inotropic P2X₇ receptor stimulates membrane internalization by a mechanism that involves intracellular GTP, but does not require internal Ca²⁺ or influx of Na⁺ or Ca²⁺ through the receptor-gated channel.     

Phosphorylation of muscle plasma membrane protein by a membrane-bound protein kinase

Protein kinase activity has been demonstrated in purified plasma membranes from rat diaphragm by measuring the incorporation of ³²P from [³²P]-ATP into endogenous membrane proteins and into histone, in vitro. Histone appears to be a better substrate than the endogenous membrane proteins; however, the properties of the enzyme are similar when phosphorylating endogenous or exogenous proteins. The activity of this membrane-associated protein kinase is not significantly affected by cyclic adenosine 3′,5′-monophosphate or by cyclic guanosine 3′,5′-monophosphate, but is inhibited by theophylline. The ³²P incorporated into membrane proteins is alkali-labile and is released from the membrane by protease digestion, but it is not removed by phospholipase C, by hydroxylamine, or by chloroform—methanoll extraction. Solubilization of ³²P-labeled membranes by sodium dodecylsulfate and fractionation by sodium dodecylsulfate polyacrylamide gel electrophoresis reveals that the radioactivity is predominantly associated with a single protein band with an apparent molecular weight of about 51 000. The phosphoprotein is a minor membrane component as judged by Coomassie blue staining.     

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.     

The Murein Hydrolase of the Bacteriophage φ3626 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 3626, 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 λ S holin in λΔSthf. 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.