Dominance in lambda S mutations and evidence for translational control

Phenotypic analysis of a collection of point mutations in the lysis gene S of bacteriophage lambda indicates that many of the S alleles exhibit at least partially dominant character, suggesting that the S gene product (gpS) must oligomerize to achieve its lethal membrane effect. Moreover, mutations found 5' to the coding sequence also show a dominant character and appear to define a site, designated sdi (structure directed initiation) where mRNA secondary structure controls the choice of initiation codons. We propose that formation of the sdi structure occludes the consensus Shine-Dalgarno sequence and results in initiation at the Met3 codon, generating a lethal 105 residue polypeptide. The model predicts that, in the absence of the sdi stem-and-loop, initiation occurs at the Met1 codon, generating a 107 residue polypeptide, which is a non-lethal inhibitor of lysis. In support of the model, alteration of the first codon was achieved using site-directed mutagenesis, resulting in an S allele that is more lethal and induces lysis significantly sooner than the wild-type.     

Dual translational initiation sites control function of the lambda S gene.

Lysis gene S of phage lambda has a 107 codon reading frame beginning with the codons Met1-Lys2-Met3. Genetic data have suggested that translational initiation occurs at both Met1 and Met3, generating two polypeptides, S107 and S105 respectively. We have proposed a model in which the proper scheduling of lysis depends on the partition of translational initiations between the two start codons. Here, using in vitro methods, we show that two stem-loop structures, one immediately upstream of the reading frame and a second approximately 10 codons within the gene, control the partitioning event. Utilizing primer-extension inhibition or 'toeprinting', we show that the two S start codons are served by two adjacent Shine-Dalgarno sequences. Moreover, the timing of lysis supported by the wild-type and a number of mutant alleles in vivo can be correlated with the ratio of ternary complex formation over Met1 and Met3 in vitro. Thus the regulation of the S gene is unique in that the products of two adjacent in-frame initiation events have opposing function.     

Charged amino-terminal amino acids affect the lethal capacity of Lambda lysis proteins S107 and S105

The lysis inhibitor protein S107 and the lysis effector protein S105 start at Met codons 1 and 3 of the Lambda S gene, respectively. The antagonistic action of both proteins precisely schedules lysis by formation of a non-specific lesion in the inner membrane through which the Lambda-encoded murein transglycosylase can pass. Here, we show that the main difference between lysis—effector and lysis—inhibitor is the degree by which an energized membrane inhibits either protein from hole formation. To dissect the structural parameters responsible for intrinsic inhibition of both proteins, charged amino acids were replaced proximal to the first putative membrane-spanning region in both S proteins. Our results show that the distribution of amino-terminal charged amino acids as well as the total amino-terminal net charge of S107 and S105 influence their lethal potential. The data are interpreted in terms of a model in which the electrostatic status of the amino-terminus of both S107 and S105 is an important feature affecting their conf or mat ional change required for formation of the S-dependent hole.     

Dual start motif in two lambdoid S genes unrelated to lambda S

The lysis gene region of phage 21 contains three overlapping reading frames, designated S21, R21, and Rz21 on the basis of the analogy with the SRRz gene cluster of phage lambda. The 71-codon S21 gene complements lambda Sam7 for lysis function but shows no detectable homology with S lambda in the amino acid or nucleotide sequence. A highly related DNA sequence from the bacteriophage PA-2 was found by computer search of the GenBank data base. Correction of this sequence by insertion of a single base revealed another 71-codon reading frame, which is accordingly designated the SPA-2 gene and is 85% identical to S21. There are thus two unrelated classes of S genes; class I, consisting of the homologous 107-codon S lambda and 108-codon P22 gene 13, and class II, consisting of the 71-codon S21 and SPA-2 genes. The codon sequence Met-Lys-(X)-Met...begins all four genes. The two Met codons in S lambda and 13 have been shown to serve as translational starts for distinct polypeptide products which have opposing functions: the shorter polypeptide serves as the lethal lysis effector, whereas the longer polypeptide acts as a lysis inhibitor. To test whether this same system exists in the class II S genes, the Met-I and Met-4 codons of S21 were altered in inducible plasmid clones and the resultant lysis profiles were monitored. Elimination of the Met-1 start results in increased toxicity, and lysis, although not complete, begins earlier, which suggests that both starts are used in the scheduling of lysis by S21 and is consistent with the idea that the 71- and 68-residue products act as a lysis inhibitor and a lysis effector, respectively. In addition, the R gene of 21 was shown to be related to P22 gene 19, which encodes a true lysozyme activity, and was also found to be nearly identical to PA-2 ORF2. We infer that the 21 and PA-2 R genes both encode lysozymes in the T4 e gene family. These three genes form a second class lambdoid R genes, with the lambda R gene being the sole member of the first class. The existence of two interchangeable but unrelated classes of S genes and R genes is discussed in terms of a model of bacteriophage evolution in which the individual gene is the unit of evolution.     

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.     

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'.     

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     

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.     

Co-expression of the Thermotoga neapolitana aglB gene with an upstream 3'-coding fragment of the malG gene improves enzymatic characteristics of recombinant AglB cyclomaltodextrinase

A cluster of Thermotoga neapolitana genes participating in starch degradation includes the malG gene of sugar transport protein and the aglB gene of cyclomaltodextrinase. The start and stop codons of these genes share a common overlapping sequence, aTGAtg. Here, we compared properties of expression products of three different constructs with aglB from T. neapolitana. The first expression vector contained the aglB gene linked to an upstream 90-bp 3'-terminal region of the malG gene with the stop codon overlapping with the start codon of aglB. The second construct included the isolated coding sequence of aglB with two tandem potential start codons. The expression product of this construct in Escherichia coli had two tandem Met residues at its N terminus and was characterized by low thermostability and high tendency to aggregate. In contrast, co-expression of aglB and the 3'-terminal region of malG (the first construct) resulted in AglB with only one N-terminal Met residue and a much higher specific activity of cyclomaltodextrinase. Moreover, the enzyme expressed by such a construct was more thermostable and less prone to aggregation. The third construct was the same as the second one except that it contained only one ATG start codon. The product of its expression had kinetic and other properties similar to those of the enzyme with only one N-terminal Met residue.     

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.     

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.     

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。     

Effect of the lambda S gene product on properties of the Escherichia coli inner membrane

The S gene of bacteriophage lambda is a late gene required for cell lysis, but unlike the other two lysis genes, R and Rz, it does not code for an endolysin. Earlier studies have shown that the S gene product inhibits respiration and macromolecular synthesis and makes the inner membrane permeable to sucrose. In this study, the effect of the S gene product on a number of Escherichia coli membrane functions (active transport, permeability, respiration, and transhydrogenase and ATPase activity) were measured, and a product of the lambda S gene was identified in the inner membrane fraction by two-dimensional polyacrylamide gel electrophoresis. The results of these experiments indicate that the lambda S product is present in the inner membrane, that it increased the permeability of the membrane for all of the small molecules that were tested, and that its action is reversible. The simplest explanation of these results is that the S gene product forms a hydrophilic pore through the inner membrane, allowing small molecules and lambda lysozyme to pass through.     

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.     

基因表达水平与同义密码子使用关系的初步研究

提出一个预测基因表达水平和同义密码子使用的自洽信息聚类方法。将同义密码子分成最适密码子、非最适密码子和稀有密码子,认为三者的使用频率是调控基因表达水平的主要因素。基于这一观点,对Ｅｃｏｌｉ和Ｙｅａｓｔ两类生物的基因表达水平和密码子的使用,用自洽信息聚类方法进行了预测。发现高低表达基因明显分开,基因表达水平被分为四级;甚高表达基因（ＶＨ）、高表达基因（Ｈ）、较低表达基因（ＬＭ）和低表达基因（ＬＬ）;     

Translational regulation of the lysis gene in RNA bacteriophage fr requires a UUG initiation codon

Summary Single nucleotide substitutions identify a UUG triplet as the initiation codon of the lysis gene in RNA bacteriophage fr. This initiation codon is non-functional in de novo initiation but is activated by translational termination at the overlapping coat gene. The UUG initiation codon is crucial for gene regulation in the phage, as it excludes uncontrolled access of ribosomes to the start of the lysis gene. Replacement of UUG by either GUG or AUG results in the loss of genetic control of the lysis gene. A model is presented in which initiation factor IF3 proofreads de novo initiation at UUG codons.     

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.