Thiol protection in membrane protein purifications: A study with phage holins

The lambda holin, or S105, is a small cytoplasmic membrane protein that controls the timing of host lysis. Using thiol-specific reagents, we determined that the single cysteine residue within S105 was heterogeneously modified during membrane extraction and subsequent immobilized metal ion chromatography. Here we describe the use of a specific and reversible thiol reagent, 2,2′-dithiodipyridine, to generate purified protein with its cysteine residues in the native thiol state. The 2,2′-dithiodipyridine protection protocol was also successfully used for another unrelated holin, S²¹68, and should be generally useful for the purification of membrane proteins.     

Overlapping genes in RNA phage: a new protein implicated in lysis.

We have identified a new 75 amino acid polypeptide (L protein) following f2 phage infection of E. coli. It is encoded by an out-of-phase overlapping gene which begins within the coat protein gene, ends in the replicase gene and covers the 36 base intercistronic space between them. A mutant f2 phage carrying a UGA mutation (op3), which complements mutations in the other three f2 genes (coat, A protein and replicase), fails to lyse cells (Model, Webster and Zinder, 1979) and also fails to produce L protein. Both lysis and L protein are restored following op3 infection of a UGA suppressor-containing strain or infection of wild-type bacteria with a revertant of op3. L protein is found in the insoluble fraction of artificially lysed cells. In this paper, we present the time course of its synthesis relative to the other f2-coded polypeptides: L protein synthesis increases as replicase synthesis decreases. We also report the discovery of another phage-coded polypeptide, which appears to be the product of a novel mode of translation: initiation at the coat protein initiation site, followed by translational frame shifting into the L protein frame and termination at the L protein terminus.     

Theoretical-experimental study of the process of phage lysis

The mathematical model of phagolytic process is proposed, taking into account the preexponential phase in the growth of the biomass of bacteria and phages. This phase seems to be connected with the existence of biological inertia in the synthesis of phage and bacterial proteins. Proceeding from the comparison with experiments on E. coli and the corresponding phage, the numerical values of the constants of the model were determined by one of the optimization methods. The comparison of theoretical calculations with the results of experimental studies showed that this mathematical model correctly described the dynamics of phagolysis both in qualitative and quantitative aspects.     

噬菌体裂解细菌过程中冷光实时监测活菌方法的建立

产毒素的霍乱弧菌Vibrio cholerae可导致严重腹泻,已引起7次全球大流行.对于烈性噬菌体清除霍乱弧菌的效果评价上,一般使用传统的活细胞培养计数及噬菌斑进行观察分析,但操作费时耗力,尤其不能实时获得菌株被裂解及残存细胞的数量变化.进一步探索简便、能够实时监测噬菌体裂解霍乱弧菌的方法是非常必要的.利用荧光报告质粒...     

Role of superinfecting phage in lysis inhibition with phage T4 in Escherichia coli

Rutberg, Blanka (Karolinska Institutet, Stockholm, Sweden), and Lars Rutberg. Role of superinfecting phage in lysis inhibition with phage T4 in Escherichia coli. J. Bacteriol. 90:891-894. 1965.-The ability of bacteriophage T4 to induce lysis inhibition upon superinfection was investigated after various treatments of the phage. This ability was found not to be a property of the external protein part of the phage, nor was it dependent on the functional and possibly structural integrity of the phage genetic material.     

Protein determinants of phage T4 lysis inhibition

Genetic studies have established that lysis inhibition in bacteriophage T4 infections occurs when the RI antiholin inhibits the lethal hole-forming function of the T holin. The T-holin is composed of a single N-terminal transmembrane domain and a ~20 kDa periplasmic domain. It accumulates harmlessly throughout the bacteriophage infection cycle until suddenly causing permeabilization of the inner membrane, thereby initiating lysis. The RI antiholin has a SAR domain that directs its secretion to the periplasm, where it can either be inactivated and degraded or be activated as a specific inhibitor of T. Previously, it was shown that the interaction of the soluble domains of these two proteins within the periplasm was necessary for lysis inhibition. We have purified and characterized the periplasmic domains of both T and RI. Both proteins were purified in a modified host that allows disulfide bond formation in the cytoplasm, due to the functional requirement of conserved disulfide bonds. Analytical centrifugation and circular dichroism spectroscopy showed that RI was monomeric and exhibited ~80% alpha-helical content. In contrast, T exhibited a propensity to oligomerize and precipitate at high concentrations. Incubation of RI with T inhibits this aggregation and results in a complex of equimolar T and RI content. Although gel filtration analysis indicated a complex mass of 45 kDa, intermediate between the predicted 30 kDa heterodimer and 60 kDa heterotetramer, sedimentation velocity analysis indicated that the predominant species is the former. These results suggest that RI binding to T is necessary and sufficient for lysis inhibition.     

Simple colorimetric microplate test of phage lysis in Salmonella enterica

A simple microplate method, based on conversion of tetrazolium to formazan, was devised for rapidly assessing Salmonella survival after phage treatment. Results were easily interpretable. Monitoring with a microplate reader was useful, but not required. The method was used in defining phage-Salmonella interactions for selection of phage biocontrol cocktails.     

Abortive phage resistance mechanism AbiZ speeds the lysis clock to cause premature lysis of phage-infected Lactococcus lactis

The conjugative plasmid pTR2030 has been used extensively to confer phage resistance in commercial Lactococcus starter cultures. The plasmid harbors a 16-kb region, flanked by insertion sequence (IS) elements, that encodes the restriction/modification system LlaI and carries an abortive infection gene, abiA. The AbiA system inhibits both prolate and small isometric phages by interfering with the early stages of phage DNA replication. However, abiA alone does not account for the full abortive activity reported for pTR2030. In this study, a 7.5-kb region positioned within the IS elements and downstream of abiA was sequenced to reveal seven additional open reading frames (ORFs). A single ORF, designated abiZ, was found to be responsible for a significant reduction in plaque size and an efficiency of plaquing (EOP) of 10(-6), without affecting phage adsorption. AbiZ causes phage phi31-infected Lactococcus lactis NCK203 to lyse 15 min early, reducing the burst size of phi31 100-fold. Thirteen of 14 phages of the P335 group were sensitive to AbiZ, through reduction in either plaque size, EOP, or both. The predicted AbiZ protein contains two predicted transmembrane helices but shows no significant DNA homologies. When the phage phi31 lysin and holin genes were cloned into the nisin-inducible shuttle vector pMSP3545, nisin induction of holin and lysin caused partial lysis of NCK203. In the presence of AbiZ, lysis occurred 30 min earlier. In holin-induced cells, membrane permeability as measured using propidium iodide was greater in the presence of AbiZ. These results suggest that AbiZ may interact cooperatively with holin to cause premature lysis.     

The effect of iodoacetate and other inhibitors on phage production and lysis in three phage systems

The minimal bacteriostatic concentration of iodoacetate, azide, or proflavine was added at intervals during the latent periods of virus in three different bacterium-bacteriophage systems (M. aureus, B. mycoides, E. coli). For each interval at which inhibitor was added, the occurrence of lysis and the final yield of phage were determined. In the B. mycoides and E. coli systems, when added during the first part of the latent period, inhibitor prevented lysis and no phage was released. Introduction of inhibitor during the last part of the latent period resulted in normal lysis and in a linear increase in phage that progressively approached the yield obtained in the absence of inhibitor (the later the introduction, the higher the yield). In the M. aureus system, phage production and lysis in the presence of inhibitor followed the same general pattern, except that release of phage and normal lysis occurred in infected cells to which inhibitor had been added quite early in the latent period. Our results, when compared with those of Foster (1948) with proflavine and Bozeman et al. (1954) with chloramphenicol, suggest that (1) the final phage yields represent the amount of mature intracellular virus present at the time of addition of inhibitor and (2) the reactions leading to lysis proceed independently of those leading to the formation of mature virus once phage infection has reached a critical point in time.     

The pinholin of lambdoid phage 21: control of lysis by membrane depolarization

The phage 21 holin, S²¹, forms small membrane holes that depolarize the membrane and is designated as a pinholin, as opposed to large-hole-forming holins, like S^λ. Pinholins require secreted SAR endolysins, a pairing that may represent an intermediate in the evolution of canonical holin-endolysin systems.     

Glucocorticoids in malignant lymphoid cells: Gene regulation and the minimum receptor fragment for lysis

We have examined clones of human malignant lymphoid cells for markers that correlate with glucocorticoid-mediated cell lysis. In glucocorticoid-sensitive clones of CEM, a human T-cell lymphoblastic leukemia line, two genes correlate with glucocorticoid-induced cell lysis. The glucocorticoid receptor (GR) itself is induced by standard glucocortoids in sensitive clones and not in insensitive clones. The phenylpyrazolo-glucocortocoid cortivazol (CVZ) is capable of lysing several clones resistant to high concentrations of standard potent glucocorticoids. When these clones were tested for cortivazol responses, they were not only lysed by cortivazol but also showed induction of GR mRNA. Thus receptor induction appears to correlate with the lysis function of receptor in these cells. To determine what parts of the GR are required for lysis, we have mapped this function by transfecting and expressing GR and GR fragment genes in a GR-deficient CEM clone. Our results indicate that none of the known trans-activation regions of the GR are required. Removal of the steroid binding domain gives a fragment that is fully constitutive. Only one and one-half “Zn fingers” of the DNA binding region are required. We also find in CEM cells rapid suppression of the c-myc protooncogene, proceding growth arrest and cell lysis by glucocorticoids. This occurs only in clones possessing both intact receptors and lysis function. Thus the simple presence of GR alone is not sufficient to guarantee c-myc down-regulation. Introduction into the cells of c-myc driven by a promoter that does not permit suppression by glucocorticoids confers resistance to steroids. Furthermore, suppression of c-myc by antisense oligonucleotides also kills the cells. Therefore, c-myc appears to be a pivotal gene related both to ability of steroid to kill and to cell viability.     

Isolation and expression of the lysis genes of Actinomyces naeslundii phage Av-1

Like most gram-positive oral bacteria, Actinomyces naeslundii is resistant to salivary lysozyme and to most other lytic enzymes. We are interested in studying the lysins of phages of this important oral bacterium as potential diagnostic and therapeutic agents. To identify the Actinomyces phage genes encoding these species-specific enzymes in Escherichia coli, we constructed a new cloning vector, pAD330, that can be used to enrich for and isolate phage holin genes, which are located adjacent to the lysin genes in most phage genomes. Cloned holin insert sequences were used to design sequencing primers to identify nearby lysin genes by using whole phage DNA as the template. From partial digestions of A. naeslundii phage Av-1 genomic DNA we were able to clone, in independent experiments, inserts that complemented the defective lambda holin in pAD330, as evidenced by extensive lysis after thermal induction. The DNA sequence of the inserts in these plasmids revealed that both contained the complete lysis region of Av-1, which is comprised of two holin-like genes, designated holA and holB, and an endolysin gene, designated lysA. We were able to subclone and express these genes and determine some of the functional properties of their gene products.     

Two mathematical models for the development of a single microbial pellet

A mathematical model for pellet development of filamentous microorganisms is presented, which simulates in detail location and growth of single hyphal elements. The basic model for growth, septation and branching of discrete hyphae is adopted from Yang et al. [2, 23]. Exact solutions to the intracellular mass-balance equations of a growth-limiting key component is given for two types of either branched or unbranched cellular compartments. Furthermore, the growth model was extended in regard to the external mass-balance equations of limiting substrates (oxygen, glucose) under the assumption that the substrates can enter the denser regions of the pellet only diffusively. Penetration of the substrates into the more porous outer regions of the pellet occurs more easily due to microeddies in the surrounding fluid. Chipping of hyphae from the pellet surface by shear forces was included in the model as well. The application of shear forces leads to a marked smoothing of the simulated pellet surface. The development of pellets from spore germination up to late stages with cell-lysis due to shortage of substrates in the pellet centre can be described. The effects of various model parameters are discussed.List of Symbols A _i algebraic coefficient (i = 1, 2,..., 6) - B _i algebraic coefficient (i = 1, 2,..., 6) - C _i mass-concentration of component i (i = O₂, S) (gl^–1) - C _i,crit concentration of substance i critical for lysis (i=O₂, S) (gl^–1) - C _i,stop concentration of substance i below which cells are inactivated (gl^–1) - C(l _i,t) intracellular concentration of the key component at site l _i and time t (gl^–1) - C _m maximal intracellular concentration of the key component (gl^–1) - C _X Concentration of dry biomass (gl^–1) - D intracellular diffusion coefficient of the key component (m² h^–1) - D _max,i maximal molecular diffusion coefficient of substrate i (i = O₂, S) (m² h^–1) - D _eff,i effective diffusion coefficient of component i (i = O₂) (m² h^–1) - d _h cross-sectional diameter of hyphae (m) - k production coefficient for the key component (h^–1) - K _s Monod coefficient for glucose (gl^–1) - k ₀ Monod coefficient for oxygen (gl^–1) - L _c total length of a compartment (m) - L _i total length of branch i (i=1, 2, 3) (m) - l _i position on branch i (i=1, 2, 3) - L _m maximal length of a segment (m) - m _i maintenance coefficient of substrate i (h^–1) - N _m maximal number of segments in a compartment - n _iR number of tips of type i in layer R, i=1, 2 - p auxiliary variable (see Eq. (7)) - P _Br probability that a hypha is chipped off (%h^–1) - pO ₂ partial pressure of oxygen in the liquid phase (%) - Q auxiliary variable (see Eq. (8)) - Q _i uptake rate of substrate i (i = O₂, S) (gl^–1 h^–1) - q auxiliary variable (see Eq. (7)) - R index of radial layer (R=1, 2, 3,..., R _max) - r radius (m) - r _crit critical radius, Eq. (15) (m) - r _max pellet radius (m) - r _tip distance from the pellet centre to the tip position (m) - r _thr threshold radius (m) - s auxiliary variable (see Eq. (7)) - S index for glucose - t time (h) - v _R volume of layer R (1) - Y _Mi observable yield coefficient of biomass on substrate i (gg^–1) - Y _Xi yield coefficient of biomass on substrate i (gg^–1) Greek Letters _i actual tip expansion rate (m h^–1) - _i,m actual maximal extension rate of tip i (i=1, 2) (m h^–1) - _1y lysis rate (h^–1) - _m maximal tip extension rate (m h^–1) - auxiliary variable in Eq. (2) - auxiliary variable in Eq. (3) - auxiliary variable defined in Eq. (4) (m^–1) - _shear shear force parameter - _R overall specific growth rate in layer R (h^–1) - _m maximal specific hyphal growth rate (h^–1) - cell volume density (l cell volume per 1) - _crit critical cell volume density in Eq. (15) - _S shear force parameter - _X cell mass density (g dry weight per 1 wet cells) - (C _i) growth kinetics on substrate i - proportional factor in Eq. (34) (l g^–1) We thank the Deutsche Forschungsgemeinschaft (DFG) for financially supporting parts of this work.We thank the Deutsche Forschungsgemeinschaft (DFG) for financially supporting parts of this work.     

Overcoming the phage replication threshold: a mathematical model with implications for phage therapy

Prior observations of phage-host systems in vitro have led to the conclusion that susceptible host cell populations must reach a critical density before phage replication can occur. Such a replication threshold density would have broad implications for the therapeutic use of phage. In this report, we demonstrate experimentally that no such replication threshold exists and explain the previous data used to support the existence of the threshold in terms of a classical model of the kinetics of colloidal particle interactions in solution. This result leads us to conclude that the frequently used measure of multiplicity of infection (MOI), computed as the ratio of the number of phage to the number of cells, is generally inappropriate for situations in which cell concentrations are less than 10(7)/ml. In its place, we propose an alternative measure, MOI(actual), that takes into account the cell concentration and adsorption time. Properties of this function are elucidated that explain the demonstrated usefulness of MOI at high cell densities, as well as some unexpected consequences at low concentrations. In addition, the concept of MOI(actual) allows us to write simple formulas for computing practical quantities, such as the number of phage sufficient to infect 99.99% of host cells at arbitrary concentrations.     

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.     

Functioning of the cloned phage MS2 lysis protein in Escherichia coli impaired in murein synthesis

Abstract The mode of action of the phage MS2 lysis protein seems not to involve a direct interaction with the murein synthesis machinery as is the case for lysis induced by β-lactam antibiotics. Mutants with defects in various penicillin-binding proteins, which are involved in murein synthesis, were found to show normal lysis sensitivity towards the cloned MS2 lysis protein. In addition, both processes, longitudinal growth of the murein sacculus in the presence of furazlocillin, cephalexin and nalidixic acid as well as spherical growth in the presence of mecillinam were sensitive to the phage lysis protein. No change in the capacity of the binding proteins to bind ¹⁴C-labelled penicillin G was observed in the presence of the MS2 lysis gene product.