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.     

Reconstitution of holin activity with a synthetic peptide containing the 1-32 sequence region of EJh,the EJ-1 phage holin

Pneumococcal EJ-1 phage holin (EJh) is a hydrophobic polypeptide of 85 amino acid residues displaying lethal inner membrane disruption activity. To get an insight into holin structure and function, several peptides representing the different topological regions predicted by sequence analysis have been synthesized. Peptides were structurally characterized in both aqueous buffer and membrane environments, and their potential to induce membrane perturbation was determined. Among them, only the N-terminal predicted transmembrane helix increased the membrane permeability. This segment, only when flanked by the positive charged residues on its N-terminal side, which are present in the sequence of the full-length protein, folds into a major alpha-helix structure with a transmembrane preferential orientation. Fluorescein quenching experiments of N-terminal-labeled peptide evidenced the formation of oligomers of variable size depending on the peptideto-lipid molar ratio. The self-assembling tendency correlated with the formation of transmembrane pores that permit the release of encapsulated dextrans of various sizes. When analyzed by atomic force microscopy, peptide-induced membrane lesions are visualized as transbilayer holes. These findings are the first evidence for a lytic domain in holins and for the nature of membrane lesions caused by them.     

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.     

Prosystemin-antimicrobial-peptide fusion reduces tomato late blight lesion expansion

Antimicrobial peptides offer a new method for controlling pathogens, however, many promising peptides are too small for direct production in plants. A protein delivery system was developed based on a proteolytic mechanism used by Solanaceous plants to produce the very small (18 amino acid) signaling peptide systemin from the polypeptide prosystemin. Fusion of the gene encoding the 23 kDa protein prosystemin with the antimicrobial peptide (pep11) sequence, replacing the systemin sequence, allowed for expression in transgenic tomato plants. Six days after inoculation with the late blight pathogen Phytophthora infestans, detached leaflets of transgenic tomato (Rutgers) exhibited a reduction in lesion size of at least 50 percent.     

Functional regulation of the Listeria monocytogenes bacteriophage A118 holin by an intragenic inhibitor lacking the first transmembrane domain

We have dissected the functional properties of the holin encoded by Listeria monocytogenes bacteriophage A118. Native hol118 was cloned into lambdaDeltaSthf, devoid of the S holin, and tested in an E. coli background. Surprisingly, it caused very late cell lysis, beginning at 80 min after induction. Immunological analyses demonstrated that Hol118 appears in the cytoplasmic membrane shortly after infection. The hol118 gene features a dual start motif similar to lambda S. Therefore, different N-terminally modified Hol118 variants were tested. However, in contrast to lambda S, inactivation of AUG-1 or AUG-2 showed no significant influence on lysis timing. In addition, Hol118-mediated lysis could not be triggered by energy poisons, indicating a functional regulation different from that of S. Toeprinting assays on hol118 mRNA revealed an unexpected translational start codon (AUG-3) at nucleotide position 40. We demonstrated by in vitro and in vivo approaches that the predicted Hol118(83) product is actually produced together with the full-length polypeptide. However, although the truncated holin lacking its first transmembrane domain appeared in the cytoplasmic membrane, it was shown to be functionally deficient and unable to support lambda R-mediated lysis. In contrast, specific mutations introduced to abolish translation initiation at AUG-3 drastically accelerated lysis, pointing to an inhibitor function of Hol118(83). This hypothesis was supported by the observation that hol118(83) inhibited holin function when expressed in trans. A deviation from the lambda S paradigm is proposed, which represents a new model of holin functional regulation: the intragenic, in frame translated Hol118(83) product, which is devoid of its first transmembrane domain, acts as a functional inhibitor and constitutes a key part of the lysis clock of A118. Presence of the dominant inhibitor function also explains the long latent period of A118, where the onset of lysis takes about 70 min, more than twice the time needed by lambda.     

Topological examination of the bacteriophage lambda S holin by EPR spectroscopy

The S protein from bacteriophage lambda is a three-helix transmembrane protein produced by the prophage which accumulates in the host membrane during late gene expression. It is responsible for the first step in lysing the host cell at the end of the viral life cycle by multimerizing together to form large pores which permeabilize the host membrane to allow the escape of virions. Several previous studies have established a model for the assembly of holin into functional holes and the manner in which they pack together, but it is still not fully understood how the very rapid transition from monomer or dimer to multimeric pore occurs with such precise timing once the requisite threshold is reached. Here, site-directed spin labeling with a nitroxide label at introduced cysteine residues is used to corroborate existing topological data from a crosslinking study of the multimerized holin by EPR spectroscopy. CW-EPR spectral lineshape analysis and power saturation data are consistent with a three-helix topology with an unstructured C-terminal domain, as well as at least one interface on transmembrane domain 1 which is exposed to the lumen of the hole, and a highly constrained steric environment suggestive of a tight helical packing interface at transmembrane domain 2.     

Genetic analysis of the T4 holin: timing and topology

The t protein of bacteriophage T4 shares with other holins the ability to cause the formation of a lethal membrane lesion which allows the phage endolysin to attack the peptidoglycan. Moreover, T, like other holins, acts in a saltatory manner at a precisely programmed time in the vegetative cycle. Unlike other holins, however, T has the unique ability to postpone its lethal function in response to a secondary infection by a T-even phage during the vegetative cycle. A signal transduction system that responds to the secondary infection is thought to be encoded by some of the numerous r genes, defined by mutations that abolish this lysis-inhibition (LIN) response. The primary structure of T differs from two main structural patterns found in more than 30 orthologous groups of holins. Genetic approaches were taken to probe the t sequence for features involved in membrane localization, functional timing and LIN regulation. Gene fusion analysis indicates that T has a single TMD near the N-terminus, with the bulk of the protein residing in the periplasm. Mapping and phenotypic analysis of deletion and point mutations in t indicates that the periplasmic domain of T is the major determinant of the timing mechanism and is involved in the LIN response.     

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.     

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.     

死亡素与泛素在大肠杆菌中的高效融合表达

死亡素是由21个氨基酸残基组成的广谱抗菌肽。为了高效表达可溶性的死亡素,本研究利用递归式PCR（recursive PCR, rPCR）扩增了死亡素基因thanatin,并将其和家蝇Musca domestica泛素基因ubiquitin构成嵌合基因,克隆到表达载体pET-32a,再与硫氧还蛋白融合后构建表达载体pET-TRX-UBI-THA。将酶切和测序鉴定正确的质粒转化表达宿主菌BL21,经0.6 mmol/L IPTG诱导,TRX-UBI-THA融合蛋白得到了高效可溶性表达。SDS-PAGE和Western blot检测结果表明融合蛋白的分子量为28.9 kD,与预期的结果一致,表达量占菌体总蛋白的46％。Western blot分析结果显示融合蛋白能与Ni-NTA鏊合物特异性的结合,表明在融合蛋白的N-端带有6×His标签。利用C-端带有6×His标签的泛素C-端水解酶对融合蛋白进行切割,切割产物经Ni²⁺-NTA亲和柱和HPLC纯化（纯化量为5.4 mg/L）,Tricince-SDS-PAGE电泳得到单一的泛素蛋白条带。电喷雾质谱(ESI-MS)分析表明,纯化的泛素分子量为2.57 kD,与通过氨基酸预测的分子量完全一致。利用琼脂孔穴扩散法对泛素活性进行检测,结果显示纯化的泛素对大肠杆菌K12D31和金黄色葡萄球菌Staphylococcus aureus具有较强的活性抑制。本研究表明,利用泛素融合技术可以高效表达可溶性的死亡素。     

Molecular function of the dual-start motif in the λ S holin

The lambda S gene represents the prototype of holin genes with a dual-start motif, which leads to the synthesis of two polypeptides, S105 and S107. They differ at their N-terminus by only two amino acids, Met-1 and Lys-2, at the beginning of the longer product. Despite the minor difference, the two proteins have opposing functions in lysis, with protein S107 being an inhibitor and protein S105 being an effector of 'hole formation' in the inner membrane. Here, we have studied the molecular mechanism underlying the 'lysis clock' contributed by the dual-start motif. We have used protein fusions in which the secretory signal sequence of the M13 procoat protein VIII has been abutted to the N-terminal Met residues of S105 and S107 respectively. S-dependent 'hole formation' required removal of the signal sequence in both fusion proteins, as both the VIII-S105 and the VIII-S107 fusion proteins were non-functional when leader peptidase cleavage was inhibited. These results strongly supported the hypothesis that functional assembly of S proteins requires translocation of their N-terminus to the periplasm. Using signal sequence cleavage as a measure of translocation, we observed that the translocation kinetics of the N-terminus of the S107 moiety was reduced about threefold when compared with the N-terminus of the S105 moiety. Moreover, depolarization of the membrane resulted in an immediate cleavage of the signal sequence and 'hole formation' exerted by the S107 moiety of the VIII-S107 fusion protein. A model is presented in which S107 with a reversed topology of its N-terminus interacts with S105 and poisons 'hole formation'. Upon depolarization of the membrane, translocation of the N-terminus of S107 to the periplasm results in the functional assembly of S proteins, i.e. 'hole formation'.     

The C-terminal sequence of the lambda holin constitutes a cytoplasmic regulatory domain

The C-terminal domains of holins are highly hydrophilic and contain clusters of consecutive basic and acidic residues, with the overall net charge predicted to be positive. The C-terminal domain of lambda S was found to be cytoplasmic, as defined by protease accessibility in spheroplasts and inverted membrane vesicles. C-terminal nonsense mutations were constructed in S and found to be lysis proficient, as long as at least one basic residue is retained at the C terminus. In general, the normal intrinsic scheduling of S function is deranged, resulting in early lysis. However, the capacity of each truncated lytic allele for inhibition by the S107 inhibitor product of S is retained. The K97am allele, when incorporated into the phage context, confers a plaque-forming defect because its early lysis significantly reduces the burst size. Finally, a C-terminal frameshift mutation was isolated as a suppressor of the even more severe early lysis defect of the mutant SA52G, which causes lysis at or before the time when the first phage particle is assembled in the cell. This mutation scrambles the C-terminal sequence of S, resulting in a predicted net charge increase of +4, and retards lysis by about 30 min, thus permitting a viable quantity of progeny to accumulate. Thus, the C-terminal domain is not involved in the formation of the lethal membrane lesion nor in the "dual-start" regulation conserved in lambdoid holins. Instead, the C-terminal sequence defines a cytoplasmic regulatory domain which affects the timing of lysis. Comparison of the C-terminal sequences of within holin families suggests that these domains have little or no structure but act as reservoirs of charged residues that interact with the membrane to effect proper lysis timing.     

Sizing particles with a Coulter counter

A theorem is presented which permits a determination of the amplitude of the signal generated by the passage of a particle of arbitrary shape through a Coulter counter. The theorem is applied to particles of two shapes, a sphere and a prolate spheroid. For the sphere the signal is directly proportional to the volume of the particle. For the spheroid the result is a complicated function of the shape. Two spheroids of the same volume but different shapes will give different signals.     

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.     

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。     

Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion

Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP. Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble-insoluble phase transition behavior imparted by the ELP tag. Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4-h culture with chemical induction of the plasmid-borne fusion protein gene and a 24-h culture without chemical induction. The total protein content and functional activity were quantified at each ITC purification step. For CAT, BFP, and Trx, the 24-h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4-h-induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion. For these proteins, ITC purification of fusion proteins also results in approximately 75% recovery of active fusion protein, similar to affinity chromatography. Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high-throughput, microscale purification; thus, potentially impacting both high-throughput protein expression and purification for proteomics and large scale, cost-effective industrial bioprocessing of pharmaceutically relevant proteins.