The mycobacteriophage Ms6 encodes a chaperone‐like protein involved in the endolysin delivery to the peptidoglycan

Like most double‐stranded (ds) DNA phages, mycobacteriophage Ms6 uses the holin‐endolysin system to achieve lysis of its host. In addition to endolysin (lysA) and holin (hol) genes, Ms6 encodes three accessory lysis proteins. In this study we investigated the lysis function of Gp1, which is encoded by the gp1 gene that lies immediately upstream of lysA. Escherichia coli lysis was observed after coexpression of LysA and Gp1 in the absence of Ms6 holin. Gp1 does not belong to the holin class of proteins, and we provide evidence that it shares several characteristics with molecular chaperones. We show that Gp1 interacts with LysA, and that this interaction is necessary for LysA delivery to its target. In addition, PhoA fusions showed that, in Mycobacterium smegmatis, LysA is exported to the extracytoplasmic environment in the presence of Gp1. We also show that Gp1 is necessary for efficient M. smegmatis lysis, as Ms6 gp1 deletion results in host lysis defects. We propose that delivery of Ms6 endolysin to the murein layer is assisted by Gp1, a chaperone‐like protein, in a holin‐independent manner.     

The Endolysin-Binding Domain Encompasses the N-Terminal Region of the Mycobacteriophage Ms6 Gp1 Chaperone

The intermolecular interactions of the mycobacteriophage Ms6 secretion chaperone with endolysin were characterized. The 384-amino-acid lysin (lysin₃₈₄)-binding domain was found to encompass the N-terminal region of Gp1, which is also essential for a lysis phenotype in Escherichia coli. In addition, a GXXXG-like motif involved in Gp1 homo-oligomerization was identified within the C-terminal region.     

A second endolysin gene is fully embedded in-frame with the lysA gene of mycobacteriophage Ms6

Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts. The mycobacteriophage Ms6 accomplishes lysis by producing two cell wall hydrolytic enzymes, Lysin A (LysA) that possesses a central peptidoglycan recognition protein (PGRP) super-family conserved domain with the amidase catalytic site, that cleaves the amide bond between the N-acetylmuramic acid and L-alanine residues in the oligopeptide crosslinking chains of the peptidoglycan and Lysin B (LysB) a mycolylarabinogalactan esterase that hydrolyzes the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex. Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA(241)) encoded in the same reading frame and preceded by a consensus ribosome-binding site. In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin(384)) or the shorter (Lysin(241)) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis. In addition, both endolysins have peptidoglycan hydrolase activity and demonstrated broad growth inhibition activity against various gram-positive bacteria and mycobacteria.     

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.     

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.     

Pinholin S21 mutations induce structural topology and conformational changes

The bacteriophage infection cycle is terminated at a predefined time to release the progeny virions via a robust lytic system composed of holin, endolysin, and spanin proteins. Holin is the timekeeper of this process. Pinholin S²¹ is a prototype holin of phage Φ21, which determines the timing of host cell lysis through the coordinated efforts of pinholin and antipinholin. However, mutations in pinholin and antipinholin play a significant role in modulating the timing of lysis depending on adverse or favorable growth conditions. Earlier studies have shown that single point mutations of pinholin S²¹ alter the cell lysis timing, a proxy for pinholin function as lysis is also dependent on other lytic proteins. In this study, continuous wave electron paramagnetic resonance (CW-EPR) power saturation and double electron-electron resonance (DEER) spectroscopic techniques were used to directly probe the effects of mutations on the structure and conformational changes of pinholin S²¹ that correlate with pinholin function. DEER and CW-EPR power saturation data clearly demonstrate that increased hydrophilicity induced by residue mutations accelerate the externalization of antipinholin transmembrane domain 1 (TMD1), while increased hydrophobicity prevents the externalization of TMD1. This altered hydrophobicity is potentially accelerating or delaying the activation of pinholin S²¹. It was also found that mutations can influence intra- or intermolecular interactions in this system, which contribute to the activation of pinholin and modulate the cell lysis timing. This could be a novel approach to analyze the mutational effects on other holin systems, as well as any other membrane protein in which mutation directly leads to structural and conformational changes.     

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.     

The murein hydrolase of the bacteriophage phi3626 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 phi3626, 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 lambda S holin in lambdadeltaSthf. 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.     

Complete Genomic and Lysis-Cassette Characterization of the Novel Phage,KBNP1315, which Infects Avian Pathogenic Escherichia coli (APEC)

Avian pathogenic Escherichia coli (APEC) is a major pathogen that causes avian colibacillosis and is associated with severe economic losses in the chicken-farming industry. Here, bacteriophage KBNP1315, infecting APEC strain KBP1315, was genomically and functionally characterized. The evolutionary relationships of KBNP1315 were analyzed at the genomic level using gene (protein)-sharing networks, the Markov clustering (MCL) algorithm, and comparative genomics. Our network analysis showed that KBNP1315 was connected to 30 members of the Autographivirinae subfamily, which comprises the SP6-, T7-, P60-, phiKMV-, GAP227- and KP34-related groups. Network decomposition suggested that KBNP1315 belongs to the SP6-like phages, but our comparison of putative encoded proteins revealed that key proteins of KBNP1315, including the tail spike protein and endolysin, had relative low levels of amino acid sequence similarity with other members of the SP6-like phages. Thus KBNP1315 may only be distantly related to the SP6-like phages, and (based on the difference in endolysin) its lysis mechanism may differ from theirs. To characterize the lytic functions of the holin and endolysin proteins from KBNP1315, we expressed these proteins individually or simultaneously in E. coli BL21 (DE3) competent cell. Interestingly, the expressed endolysin was secreted into the periplasm and caused a high degree of host cell lysis that was dose-dependently delayed/blocked by NaN₃-mediated inhibition of the SecA pathway. The expressed holin triggered only a moderate inhibition of cell growth, whereas coexpression of holin and endolysin enhanced the lytic effect of endolysin. Together, these results revealed that KBNP1315 appears to use a pin-holin/signal-arrest-release (SAR) endolysin pathway to trigger host cell lysis.     

Subclassification and targeted characterization of prophage-encoded two-component cell lysis cassette

Bacteriophage induced lysis of host bacterial cell is mediated by a two component cell lysis cassette comprised of holin and lysozyme. Prophages are integrated forms of bacteriophages in bacterial genomes providing a repertoire for bacterial evolution. Analysis using the prophage database (http://bicmku.in:8082) constructed by us showed 47 prophages were associated with putative two component cell lysis genes. These proteins cluster into four different subgroups. In this process, a putative holin (essd) and endolysin (ybcS), encoded by the defective lambdoid prophage DLP12 was found to be similar to two component cell lysis genes in functional bacteriophages like p21 and P1. The holin essd was found to have a characteristic dual start motif with two transmembrane regions and C-terminal charged residues as in class II holins. Expression of a fusion construct of essd in Escherichia coli showed slow growth. However, under appropriate conditions, this protein could be over expressed and purified for structure function studies.The second component of the cell lysis cassette, ybcS, was found to have an N-terminal SAR (Signal Arrest Release) transmembrane domain. The construct of ybcS has been over expressed in E.coli and the purified protein was functional, exhibiting lytic activity against E.coli and Salmonella typhi cell wall substrate. Such targeted sequence- structure-function characterization of proteins encoded by cryptic prophages will help understand the contribution of prophage proteins to bacterial evolution.     

The Lysis System of the <Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> Phage μ1/6

Previously, two genes, designated as lyt and hol, were identified in the lysis module of phage μ1/6. They were cloned and expressed in Escherichia coli. An additional candidate holin gene, hol2, was found downstream from the hol gene based on one predicted transmembrane domain and a highly charged C-terminal sequence of the encoded protein. Expression of hol or hol2 in E. coli was shown to cause cell death. The concomitant expression of λ endolysin (R) and μ1/6 holin resulted in cell lysis. Similarly, the coexpression of the endolysin and holin of phage μ1/6 led to lysis, apparently due to the ability of μ1/6 endolysin to hydrolyze the peptidoglycan layer of this bacterium. In contrast, the simultaneous expression of μ1/6 hol2 and the endolysin gene (λR or μ1/6 lyt) did not cause detectable lysis of the host cells. Demonstration of the holin function in streptomycetes was achieved by providing for the release of μ1/6 endolysin to the periplasm and subsequent cleavage of the peptidoglycan, which strongly suggested that the holin produces lesions in the streptomycete membrane.     

Identification of a holin encoded by the<Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> phage µ1/6; functional analysis in<Emphasis Type="Italic">Escherichia coli</Emphasis> system

An open reading frame encoding an 88 amino acid protein was present downstream of the previously characterized endolysin ofStreptomyces aureofaciens phage μ1/6. Structural analysis of its sequence revealed features characteristic for holin. This open reading frame encoding the putative holin was amplified by polymerase chain reaction and cloned into the expression vector pET-21d(+). Synthesis of the holin-like protein resulted in bacterial cell death but not lysis. Theholμ1/6 gene was able to complement the defective λS allele in the nonsuppressingEscherichia coli HB101 strain to produce phage progeny. This fact suggests that the proteins encoded by both phage genes have analogous function,i.e. the streptomycete holin induces nonspecific lesions in the cytoplasmic membrane, through which the λ endolysin gains an access to its substrate, the cell wall. The concomitant expression of bothS. aureofaciens holμ1/6 and λ endolysin inE. coli resulted in abrupt cell lysis. This result provided further evidence that the product ofholμ1/6 gene is a holin. This work was supported by the VEGA grant of theSlovak Academy of Sciences no. 2/5070/25 and grant of theMinistry of Agriculture of the Slovak Republic no. 2003 5P27/0208 E02.     

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 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.     

Lysis and lysis inhibition in bacteriophage T4: rV mutations reside in the holin t gene.

Upon infecting populations of susceptible host cells, T-even bacteriophages maximize their yield by switching from lysis at about 25 to 35 min at 37 degrees C after infection by a single phage particle to long-delayed lysis (lysis inhibition) under conditions of sequential infection occurring when free phages outnumber host cells. The timing of lysis depends upon gene t and upon one or more rapid-lysis (r) genes whose inactivation prevents lysis inhibition. t encodes a holin that mediates the movement of the T4 endolysin though the inner cell membrane to its target, the cell wall. The rI protein has been proposed to sense superinfection. Of the five reasonably well characterized r genes, only two, rI and rV, are clearly obligatory for lysis inhibition. We show here that rV mutations are alleles of t that probably render the t protein unable to respond to the lysis inhibition signal. The tr alleles cluster in the 5' third of t and produce a strong r phenotype, whereas conditional-lethal t alleles produce the classical t phenotype (inability to lyse) and other t alleles produce additional, still poorly understood phenotypes. tr mutations are dominant to t+, a result that suggests specific ways to probe T4 holin function.     

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.     

Three functions of bacteriophage P1 involved in cell lysis.

Amber and deletion mutants were used to assign functions in cell lysis to three late genes of bacteriophage P1. Two of these genes, lydA and lydB of the dar operon, are 330 and 444 bp in length, respectively, with the stop codon of lydA overlapping the start codon of lydB. The third, gene 17, is 558 bp in length and is located in an otherwise uncharacterized operon. A search with the predicted amino acid sequence of LydA for secondary motifs revealed a holin protein-like structure. Comparison of the deduced amino acid sequence of gene 17 with sequences of proteins in the SwissProt database revealed homologies with the proteins of the T4 lysozyme family. The sequence of lydB is novel and exhibited no known extended homology. To study the effect of gp17, LydA, and LydB in vivo, their genes were cloned in a single operon under the control of the inducible T7 promoter, resulting in plasmid pAW1440. A second plasmid, pAW1442, is identical to pAW1440 but has lydB deleted. Induction of the T7 promoter resulted in a rapid lysis of cells harboring pAW1442. In contrast, cells harboring pAW1440 revealed only a small decrease in optical density at 600 nm compared with cells harboring vector alone. The rapid lysis phenotype in the absence of active LydB suggests that this novel protein might be an antagonist of the holin LydA.     

Endolysin of bacteriophage BFK20: evidence of a catalytic and a cell wall binding domain

A gene product of ORF24' was identified on the genome of corynephage BFK20 as a putative phage endolysin. The protein of endolysin BFK20 (gp24') has a modular structure consisting of an N-terminal amidase_2 domain (gp24CD) and a C-terminal cell wall binding domain (gp24BD). The C-terminal domain is unrelated to any of the known cell wall binding domains of phage endolysins. The whole endolysin gene and the sequences of its N-terminal and C-terminal domains were cloned; proteins were expressed in Escherichia coli and purified to homogeneity. The lytic activities of endolysin and its catalytic domain were demonstrated on corynebacteria and bacillus substrates. The binding activity of cell wall binding domain alone and in fusion with green fluorescent protein (gp24BD-GFP) were shown by specific binding assays to the cell surface of BFK20 host Brevibacterium flavum CCM 251 as well as those of other corynebacteria.     

The bacteriophage T4 rapid-lysis genes and their mutational proclivities

Like most phages with double-stranded DNA, phage T4 exits the infected host cell by a lytic process requiring, at a minimum, an endolysin and a holin. Unlike most phages, T4 can sense superinfection (which signals the depletion of uninfected host cells) and responds by delaying lysis and achieving an order-of-magnitude increase in burst size using a mechanism called lysis inhibition (LIN). T4 r mutants, which are unable to conduct LIN, produce distinctly large, sharp-edged plaques. The discovery of r mutants was key to the foundations of molecular biology, in particular to discovering and characterizing genetic recombination in T4, to redefining the nature of the gene, and to exploring the mutation process at the nucleotide level of resolution. A number of r genes have been described in the past 7 decades with various degrees of clarity. Here we describe an extensive and perhaps saturating search for T4 r genes and relate the corresponding mutational spectra to the often imperfectly known physiologies of the proteins encoded by these genes. Focusing on r genes whose mutant phenotypes are largely independent of the host cell, the genes are rI (which seems to sense superinfection and signal the holin to delay lysis), rIII (of poorly defined function), rIV (same as sp and also of poorly defined function), and rV (same as t, the holin gene). We did not identify any mutations that might correspond to a putative rVI gene, and we did not focus on the famous rII genes because they appear to affect lysis only indirectly.