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PubMed1995

A new procedure for efficient recovery of DNA, RNA, and proteins from Listeria cells by rapid lysis with a recombinant bacteriophage endolysin.

A method for the rapid lysis of Listeria cells, employing a recombinant Listeria bacteriophage A118 lytic enzyme (PLY118), is described. The procedure can be used with all listerial species. It enables fast, efficient, and gentle recovery of DNA, RNA, or native cellular proteins from small-scale (2- to 5-ml) cultures. Moreover, this approach should be very useful in analytical detection and differentiation of Listeria strains when the release of native nucleic acids or proteins is required.

Bacterial Proteins↗
PubMed2000

Holins: the protein clocks of bacteriophage infections.

Two proteins, an endolysin and a holin, are essential for host lysis by bacteriophage. Endolysin is the term for muralytic enzymes that degrade the cell wall; endolysins accumulate in the cytosol fully folded during the vegetative cycle. Holins are small membrane proteins that accumulate in the membrane until, at a specific time that is "programmed" into the holin gene, the membrane suddenly becomes permeabilized to the fully folded endolysin. Destruction of the murein and bursting of the cell are immediate sequelae. Holins control the length of the infective cycle for lytic phages and so are subject to intense evolutionary pressure to achieve lysis at an optimal time. Holins are regulated by protein inhibitors of several different kinds. Holins constitute one of the most diverse functional groups, with >100 known or putative holin sequences, which form >30 ortholog groups.

Amino Acid Sequence↗
PubMed1995

Holins: form and function in bacteriophage lysis.

During the lytic cycle of most bacteriophages, a phage-encoded peptidoglycan-degrading activity is elaborated. At least four entirely distinct types of enzymes fulfill this role and are given the generic name 'endolysin'. Endolysins characterized to date are synthesized without a signal sequence and thus accumulate fully folded and active in the cytosol during the vegetative phase. Small membrane proteins are required in order for endolysins to gain access to the peptidoglycan. Because the available data suggest that the membrane lesion formed by these proteins is stable and non-specific, these proteins have been given the designation 'holins' ('hole'-formers). Analysis of the primary sequence suggests a simple membrane topology with two or more membrane-spanning helical domains and a highly charged, hydrophilic C-terminus. Comparison of the sequences of holins from phages of Gram-negative hosts suggests there are at least two major holin groups. Putative holin genes have also been found in bacteriophages of Gram-positive bacteria. Altogether, in phages of Eubacteria, 11 or more unrelated gene families which share the functional and structural characteristics of holins have been identified. Genetic and physiological analysis suggest that holins are primarily regulated at the level of function. Holin function is modulated in some cases by a second protein encoded by the holin gene. The primary regulation of holin function, however, appears to be intrinsic to the holin structure itself, since a missense allele of the S holin gene of phage lambda has been found which abolishes the normal delay that allows the vegetative phase to generate a useful number of progeny.

Amino Acid Sequence↗
PubMed1998

Lysis genes of the Bacillus subtilis defective prophage PBSX.

Four genes identified within the late operon of PBSX show characteristics expected of a host cell lysis system; they are xepA, encoding an exported protein; xhlA, encoding a putative membrane-associated protein; xhlB, encoding a putative holin; and xlyA, encoding a putative endolysin. In this work, we have assessed the contribution of each gene to host cell lysis by expressing the four genes in different combinations under the control of their natural promoter located on the chromosome of Bacillus subtilis 168. The results show that xepA is unlikely to be involved in host cell lysis. Expression of both xhlA and xhlB is necessary to effect host cell lysis of B. subtilis. Expression of xhlB (encoding the putative holin) together with xlyA (encoding the endolysin) cannot effect cell lysis, indicating that the PBSX lysis system differs from those identified in the phages of gram-negative bacteria. Since host cell lysis can be achieved when xlyA is inactivated, it is probable that PBSX encodes a second endolysin activity which also uses XhlA and XhlB for export from the cell. The chromosome-based expression system developed in this study to investigate the functions of the PBSX lysis genes should be a valuable tool for the analysis of other host cell lysis systems and for expression and functional analysis of other lethal gene products in gram-positive bacteria.

Bacillus Phages↗
PubMed2000

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.

Alleles↗
PubMed1965

Factors in lysis and lysis inhibition by lambda bacteriophage.

Groman, Neal B. (University of Washington, Seattle). Factors in lysis and lysis inhibition by lambda bacteriophage. J. Bacteriol. 90:1563-1568. 1965.-Induced Escherichia coli strain K-12(lambda112) exhibited lysis inhibition at 37 C but lysed at 44 C when incubated in LB medium lacking NaCl [LB - (NaCl)]. In LB medium containing NaCl, the temperatures for lysis and lysis inhibition were reversed. In contrast, induced K-12(lambda) lysed under all of these conditions. At 37 C, the addition of NaCl to LB - (NaCl) at various times after induction of K-12(lambda112) restored lysis. The degree of lysis decreased the longer the addition was delayed, but partial restoration occurred as late as 150 min postinduction. At 44 C, the addition of salt at various times after induction restored lysis inhibition even after lysis had begun. An attempt was made to correlate the conditions for lysis and lysis inhibition with the behavior of lambda112 endloysin. The enzymatic activities of lambda and mutant lambda112 endolysins were compared under various salt-temperature conditions. Both endolysins were progressively and equally inhibited by increasing concentrations of Na(+), K(+), and Li(+) salts, and exhibited similar relative activities at 24 and 37 C. Both were stable at 37 C in the presence and absence of NaCl, and were inactivated at comparable rates at 44 C. The results indicate that the effect of salt and temperature on lysis and lysis inhibition cannot be explained by their direct effect on lambda112 endolysin.

Bacteriolysis↗
PubMed1967

Lysis inhibition in Escherichia coli infected with bacteriophage T4.

A technique of continuous filtration of T4-infected Escherichia coli has been devised to study the phenomenon of lysis inhibition. Studies using this technique revealed that the length of the lysis delay caused by superinfection can attain only certain discrete values, which for low average multiplicity of superinfection is thought to be a reflection of the actual number of superinfecting particles per cell. The time interval between primary and superinfection had little effect on the length of lysis delay. With increasing rate of superinfection, the length of lysis delay decreased. In superinfected cells, the concentration of endolysin exceeded the final concentration in nonsuperinfected cells. Superinfection of a lysing culture induced lysis inhibition immediately. Temperature-shift experiments, with cells primarily infected by a temperature-sensitive endolysin mutant, revealed that after the normal latent period superinfection was unable to induce lysis inhibition. Amber-restrictive cells, which were primarily infected by an endolysin negative amber mutant, released adenosine triphosphate (ATP) at the end of the normal latent period although lysis did not occur. Superinfection reduced the loss of ATP markedly. The hypothetical role of the cytoplasmic membrane in lysis inhibition is discussed.

Adenosine Triphosphate↗
PubMed1996

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 lysiseffector, 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.

Amino Acid Sequence↗
PubMed2002

C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates.

Listeria monocytogenes phage endolysins Ply118 and Ply500 share a unique enzymatic activity and specifically hydrolyse Listeria cells at the completion of virus multiplication in order to release progeny phage. With the aim of determining the molecular basis for the lytic specificity of these enzymes, we have elucidated their domain structure and examined the function of their unrelated and unique C-terminal cell wall binding domains (CBDs). Analysis of deletion mutants showed that both domains are needed for lytic activity. Fusions of CBDs with green fluorescent protein (GFP) demonstrated that the C-terminal 140 amino acids of Ply500 and the C-terminal 182 residues of Ply118 are necessary and sufficient to direct the murein hydrolases to the bacterial cell wall. CBD500 was able to target GFP to the surface of Listeria cells belonging to serovar groups 4, 5 and 6, resulting in an even staining of the entire cell surface. In contrast, the CBD118 hybrid bound to a ligand predominantly present at septal regions and cell poles, but only on cells of serovars 1/2, 3 and 7. Non-covalent binding to surface carbohydrate ligands occurred in a rapid, saturation-dependent manner. We measured 4 x 104 and 8 x 104 binding sites for CBD118 and CBD500 respectively. Surface plasmon resonance analysis revealed unexpected high molecular affinity constants for the CBD-ligand interactions, corresponding to nanomolar affinities. In conclusion, we show that the CBDs are responsible for targeting the phage endolysins to their substrates and function to confer recognition specificity on the proteins. As the CBD sequences contain no repeats and lack all known sequence motifs for anchoring of proteins to the bacterial cell, we conclude that they use unique structural motifs for specific association with the surface of Gram-positive bacteria.

Bacteriophages↗
PubMed2002

Characterization of a holin (HolNU3-1) in methicillin-resistant Staphylococcus aureus host.

The gene encoding holin protein HolNU3-1 from a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) NU3-1 was cloned and expressed in S. aureus RN4220. HolNU3-1 encoded by the holNU3-1 gene, which is located upstream of the deleted endolysin gene, was functional. Expression of the holNU3-1 gene induced a decrease in culture turbidity and formation of translucent (empty ghost) cells in S. aureus. We found heterogeneity of the holin genes and diversity of the two-component lysis system, which consists of holin and endolysin, in MRSA hosts. We suggest that this diversity is important in the identification of the evolution of clinical isolates of S. aureus.

Amino Acid Sequence↗