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During the final step of the bacteriophage infection cycle, the cytoplasmic membrane of host cells is disrupted by small membrane proteins called holins. The function of holins in cell lysis is carried out by forming a highly ordered structure called lethal lesion, in which the accumulation of holins in the cytoplasmic membrane leads to the sudden opening of a hole in the middle of this oligomer. Previous studies showed that dimerization of holins is a necessary step to induce their higher order assembly. However, the molecular mechanism underlying the holin-mediated lesion formation is not well understood. In order to elucidate the functions of holin, we first computationally constructed a structural model for our testing system: the holin S105 from bacteriophage lambda. All atom molecular dynamic simulations were further applied to refine its structure and study its dynamics as well as interaction in lipid bilayer. Additional simulations on association between two holins provide supportive evidence to the argument that the C-terminal region of holin plays a critical role in regulating the dimerization. In detail, we found that the adhesion of specific nonpolar residues in transmembrane domain 3 (TMD3) in a polar environment serves as the driven force of dimerization. Our study therefore brings insights to the design of binding interfaces between holins, which can be potentially used to modulate the dynamics of lesion formation.

Listeria monocytogenes is an important foodborne pathogen that is a serious threat to public health security, and new strategies to control this bacterium in food are needed. HolGH15, derived from ...

Holins are small transmembrane proteins involved in the final stage of the lytic cycle of double-stranded DNA (dsDNA) phages. They cooperate with endolysins to achieve bacterial lysis, thereby releasing the phage progeny into the extracellular environment. Besides their role as membrane permeabilizers, allowing endolysin transfer and/or activation, holins also regulate the lysis timing. In this work, we provide functional characterization of the holins encoded by three phages targeting the Bacillus cereus group. The siphovirus Deep-Purple has a lysis cassette in which holP30 and holP33 encode two proteins displaying holin properties, including a transmembrane domain. The holin genes were expressed in Escherichia coli and induced bacterial lysis, with HolP30 being more toxic than HolP33. In Bacillus thuringiensis, the simultaneous expression of both holins was necessary to observe lysis, suggesting that they may interact to form functional pores. The myoviruses Deep-Blue and Vp4 both encode a single candidate holin (HolB and HolV, respectively) with two transmembrane domains, whose genes are not located near the endolysin genes. Their function as holin proteins was confirmed as their expression in E. coli impaired cell growth and viability. The HolV expression in B. thuringiensis also led to bacterial lysis, which was enhanced by coexpressing the holin with its cognate endolysin. Despite similar organizations and predicted topologies, truncated mutants of the HolB and HolV proteins showed different toxicity levels, suggesting that differences in amino acid composition influence their lysis properties.

The bacteriophage infection cycle plays a crucial role in recycling the world's biomass. Bacteriophages devise various cell lysis systems to strictly control the length of the infection cycle for an efficient phage life cycle. Phages evolved with lysis protein systems, which can control and fine-tune the length of this infection cycle depending on the host and growing environment. Among these lysis proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time and concentration hence known as the simplest molecular clock. Pinholin S21 is the holin from phage Φ21, which defines the cell lysis time through a predefined ratio of active pinholin and antipinholin (inactive form of pinholin). Active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously we reported the structural dynamics and topology of active pinholin S2168. Currently, there is no detailed structural study of the antipinholin using biophysical techniques. In this study, the structural dynamics and topology of antipinholin S2168IRS in DMPC proteoliposomes is investigated using electron paramagnetic resonance (EPR) spectroscopic techniques. Continuous-wave (CW) EPR line shape analysis experiments of 35 different R1 side chains of S2168IRS indicated restricted mobility of the transmembrane domains (TMDs), which were predicted to be inside the lipid bilayer when compared to the N- and C-termini R1 side chains. In addition, the R1 accessibility test performed on 24 residues using the CW-EPR power saturation experiment indicated that TMD1 and TMD2 of S2168IRS were incorporated into the lipid bilayer where N- and C-termini were located outside of the lipid bilayer. Based on this study, a tentative model of S2168IRS is proposed where both TMDs remain incorporated into the lipid bilayer and N- and C-termini are located outside of the lipid bilayer. This work will pave the way for the further studies of other holins using biophysical techniques and will give structural insights into these biological clocks in molecular detail.

Most endolysins of dsDNA phages are exported by a holin-dependent mechanism, while in some cases endolysins are exported via a holin-independent mechanism. However, it is still unclear whether the same endolysins can be exported by both holin-dependent and holin-independent mechanisms. This study investigated the lysis system of OP2-like phage X2 infecting Xanthomonas oryzae pv. oryzae, causing devastating bacterial leaf blight disease in rice. Based on bioinformatics and protein biochemistry methods, we show that phage X2 employs the classic "holin-endolysin" lysis system. The endolysin acts on the cell envelope and exhibits antibacterial effects in vitro, while the holin facilitates the release of the protein into the periplasm. We also characterized the role of the transmembrane domain (TMD) in the translocation of the endolysin across the inner membrane. We found that the TMD facilitated the translocation of the endolysin via the Sec secretion system. The holin increases the efficiency of protein release, leading to faster and more efficient lysis. Interestingly, in E. coli, the expression of either holin or endolysin with TMDs resulted in the formation of long rod shaped cells. We conclude that the TMD of X2-Lys plays a dual role: One is the transmembrane transport while the other is the inhibition of cell division, resulting in larger cells and thus in a higher number of released viruses per cell.

Holins are bacteriophage-encoded small transmembrane proteins that determine the phage infection cycle duration by forming nonspecific holes in the host cell membrane at a specific time postinfection. Thus, holins are also termed “protein clocks.” Holins have one or more transmembrane domains, and a charged C-terminal region, which, although conserved among holins, has not yet been examined in detail. Here, we characterize the molecular properties of the mycobacteriophage D29 holin C-terminal region, and investigate the significance of the charged residues and coiled-coil (CC) domain present therein. We show that the CC domain is indispensable for holin-mediated efficient bacterial cell lysis. We further demonstrate that, out of the positively- and negatively-charged residues present in the C-terminal region, substituting the former, and not the latter, with serine, renders holin nontoxic. Moreover, the basic residues present between the 59th and the 79th amino acids are the most crucial for holin-mediated toxicity. We also constructed an engineered holin, HolHC, by duplicating the C-terminal region. Compared to the wild type, the HolHC protein shows higher toxicity in both Escherichia coli and Mycobacterium smegmatis and causes rapid killing of both bacteria upon expression. An oligomerization property of HolHC similar to that of the wild-type holin allows us to propose that the C-terminal region of the D29 holin determines the timing, and not the extent, of oligomerization and, thereby, hole formation. Such knowledge-based engineering of mycobacteriophage holin will help in developing novel phage-based therapeutics to kill pathogenic mycobacteria, including Mycobacterium tuberculosis. IMPORTANCE Holins are bacteriophage-encoded small membrane perforators that play an important role in determining the timing of host cell lysis toward the end of the phage infection cycle. The ability of holin to precisely time the hole formation in the cell membrane, ensuring cell lysis, is both interesting and intriguing. Here, we examined the molecular properties of the mycobacteriophage D29 holin C-terminal region, which harbors several polar charged residues and a coiled-coil domain. Our data allowed us to engineer holin with an ability to rapidly kill bacteria and to show higher toxicity than the wild-type protein. Due to their ability to kill host bacteria by membrane disruption, it becomes important to explore the molecular properties of holins that allow them to function in a timely and efficient manner. Understanding these details can help us modulate holin activity and engineer bacteriophages with superior lytic properties to kill pathogenic bacteria, curtail infections, and combat antimicrobial resistance.

Prophages are widely present in Lactococcus lactis, a lactic acid bacterium (LAB) that plays a key role in dairy fermentations. L. lactis MG1363 is a laboratory strain used worldwide as a model LAB. Initially regarded as plasmid and prophage free, MG1363 carries two complete prophages, TP712 and MG-3. Only TP712 seems to be inducible but unable to lyse the host. Several so-called TP712 lysogens able to lyse upon prophage induction were reported in the past, but the reason for their lytic phenotype remained unknown. In this work, we describe CAP, a new P335 prophage detected in the “lytic TP712 lysogens” which had remained unnoticed. CAP is able to be excised after mitomycin C treatment, along with TP712, and able to infect L. lactis MG1363-like strains but not the lytic TP712 lysogens. Both phages cooperate for efficient host lysis. While the expression in trans of the CAP lytic genes was sufficient to trigger cell lysis, this process was boosted when the resident TP712 prophage was concomitantly induced. Introduction of mutations into the TP712 lytic genes revealed that its holin but not its endolysin plays a major role. Accordingly, it is shown that the lytic activity of the recombinant CAP endolysin relies on membrane depolarization. Revisiting the seminal work that generated the extensively used L. lactis MG1363 strain led us to conclude that the CAP phage was originally present in its ancestor, L. lactis NCDO712, and our results solved long-standing mysteries around the MG1363 resident prophage TP712 reported in the “presequencing” era. IMPORTANCE Prophages are bacterial viruses that integrate into the chromosomes of bacteria until an environmental trigger induces their lytic cycle, ending with lysis of the host. Prophages present in dairy starters can compromise milk fermentation and represent a serious threat in dairy plants. In this work, we discovered that two temperate phages, TP712 and CAP, infecting the laboratory strain Lactococcus lactis MG1363 join forces to lyse the host. Based on the in vitro lytic activity of the LysCAP endolysin, in combination with mutated versions of TP712 lacking either its holin or endolysin, we conclude that this cooperation relies on the combined activity of the holins of both phages that boost the activity of LysCAP. The presence of an additional prophage explains the lytic phenotype of the lysogens formerly thought to be single TP712 lysogens that had remained a mystery for many years.

Vibrio alginolyticus is a common opportunistic pathogen that can cause vibriosis of marine aquatic animals. The application of phages or particularly associated protein products for the treatment of vibriosis has shown prominent advantages compared with the treatment with traditional antibiotics. In this study, the function of a holin-endolysin system from V. alginolyticus phage HH109 was characterized by examining the effect of their overexpression on Escherichia coli and V. alginolyticus. Our data revealed that the endolysin of the phage HH109 has stronger bactericidal activity than the holin, as evidenced by observing more cell death and severe structural damage of cells in the endolysin-expressing E. coli. Furthermore, the two proteins displayed the synergistic effect when the holA and lysin were co-expressed in E. coli, although no interaction between them was detected using the bacterial two-hybrid assay. Transmission electron microscopy observation revealed disruptions of cell envelopes accompanied by leakage of intracellular contents. Similarly, the bactericidal activity of the holin and endolysin against V. alginolyticus was also examined whatever the host is sensitive or resistant to phage HH109. Together, our study contributes to a better understanding of the mechanism of phage HH109 destroying the bacterial cell wall to lyse their host and may offer alternative applications potentially for vibriosis treatment.

Large clostridial toxins (LCTs) are secreted virulence factors found in several species, including Clostridioides difficile, Clostridium perfringens, Paeniclostridium sordellii, and Clostridium novyi. LCTs are large toxins that lack a secretion signal sequence, and studies by others have shown that the LCTs of C. difficile, TcdA and TcdB, require a holin-like protein, TcdE, for secretion. The TcdE gene is located on the pathogenicity locus (PaLoc) of C. difficile, and holin-encoding genes are also present in the LCT-encoded PaLocs from P. sordellii and C. perfringens. However, the holin (TpeE) associated with the C. perfringens LCT TpeL has no homology and a different membrane topology than TcdE. In addition, TpeE has a membrane topology identical to that of the TatA protein, which is the core of the twin-arginine translocation (Tat) secretion system. To determine if TpeE was necessary and sufficient to secrete TpeL, the genes from a type C strain of C. perfringens were expressed in a type A strain of C. perfringens, HN13, and secretion was measured using Western blot methods. We found that TpeE was required for TpeL secretion and that secretion was not due to cell lysis. Mutant forms of TpeE lacking an amphipathic helix and a charged C-terminal domain failed to secrete TpeL, and mutations that deleted conserved LCT domains in TpeL indicated that only the full-length protein could be secreted. In summary, we have identified a novel family of holin-like proteins that can function, in some cases, as a system of protein secretion for proteins that need to fold in the cytoplasm. IMPORTANCE Little is known about the mechanism by which LCTs are secreted. Since LCTs are major virulence factors in clostridial pathogens, we wanted to define the mechanism by which an LCT in C. perfringens, TpeL, is secreted by a protein (TpeE) lacking homology to previously described secretion-associated holins. We discovered that TpeE is a member of a widely dispersed class of holin proteins, and TpeE is necessary for the secretion of TpeL. TpeE bears a high degree of similarity in membrane topology to TatA proteins, which form the pore through which Tat secretion substrates pass through the cytoplasmic membrane. Thus, the TpeE-TpeL secretion system may be a model for understanding not only holin-dependent secretion but also how TatA proteins function in the secretion process.

Gram-negative bacteria have evolved numerous pathways to secrete proteins across their complex cell envelopes. Here, we describe a protein secretion system which uses a holin membrane protein in tandem with a cell wall editing enzyme to mediate the secretion of substrate proteins from the periplasm to the cell exterior. The identity of the cell wall editing enzymes employed was found to vary across biological systems. For instance, the chitinase secretion pathway of Serratia marcescens uses an endopeptidase to facilitate secretion, whereas the secretion of Typhoid toxin in Salmonella enterica serovar Typhi relies on a muramidase. Various families of holins are also predicted to be involved. Genomic analysis indicates that this pathway is conserved and implicated in the secretion of hydrolytic enzymes and toxins for a range of bacteria. The pairing of holins from different families with various types of peptidoglycan hydrolases suggests that this secretion pathway evolved multiple times. We suggest that the complementary bodies of evidence presented is sufficient to propose that the pathway be named the Type 10 Secretion System (TXSS). Potential mechanisms for secretion across the outer membrane are discussed. Special Issue: Protein Secretion and Transport

P1 is a model temperate myovirus. It infects different Enterobacteriaceae and can develop lytically or form lysogens. Only some P1 adaptation strategies to propagate in different hosts are known. An atypical feature of P1 is the number and organization of cell lysis-associated genes. In addition to SAR-endolysin Lyz, holin LydA, and antiholin LydB, P1 encodes other predicted holins, LydC and LydD. LydD is encoded by the same operon as Lyz, LydA and LydB are encoded by an unlinked operon, and LydC is encoded by an operon preceding the lydA gene. By analyzing the phenotypes of P1 mutants in known or predicted holin genes, we show that all the products of these genes cooperate with the P1 SAR-endolysin in cell lysis and that LydD is a pinholin. The contributions of holins/pinholins to cell lysis by P1 appear to vary depending on the host of P1 and the bacterial growth conditions. The pattern of morphological transitions characteristic of SAR-endolysin–pinholin action dominates during lysis by wild-type P1, but in the case of lydC lydD mutant it changes to that characteristic of classical endolysin-pinholin action. We postulate that the complex lytic system facilitates P1 adaptation to various hosts and their growth conditions.

In the era of antibiotic resistance, phage therapy is gaining attention for the treatment of pathogenic organisms such as Mycobacterium tuberculosis. The selection of phages for therapeutic purposes depends upon several factors such as the host range that a phage can infect, which can be narrow or broad, time required for the host cell lysis, and the burst size. Mycobacteriophage D29 is a virulent phage that has the ability to infect and kill several slow- and fast-growing mycobacterial species including the pathogenic M. tuberculosis. It, therefore, has the potential to be used in phage therapy against M. tuberculosis. D29 lytic cassette encodes three proteins viz. peptidoglycan hydrolase (LysA), mycolylarabinogalactan esterase (LysB), and holin, which together ensure host cell lysis in a timely manner. In this work, we have scrutinized the importance of holin in mycobacteriophage D29 physiology. Bacteriophage Recombineering of Electroporated DNA (BRED) approach was used to generate D29 holin knockout (D29Δgp11), which was further confirmed by the Deletion amplification detection assay (DADA)-PCR. Our results show that D29Δgp11 is viable and retains plaque-forming ability, although with reduced plaque size. Additionally, the host cell lysis governed by the mutant phage is significantly delayed as compared to the wild-type D29. In the absence of holin, D29 shows increased latent period and reduced burst size. Thus, our experiments show that while holin is dispensable for phage viability, it is essential for the optimal phage-mediated host cell lysis and phage propagation, which further points to the significance of the “clock” function of holin. Taken together, we show the importance of holin in governing timely and efficient host cell lysis for efficient progeny phage release, which further dictates its critical role in phage biology.

Background Bacterial ghosts (BGs) are empty bacterial cell envelopes generated by releasing the cellular contents. In this study, a phage infecting Lactobacillus casei ATCC 393 (L. casei 393) was isolated and designated Lcb. We aimed at using L. casei 393 as an antigen delivery system to express phage-derived holin for development of BGs. Results A gene fragment encoding holin of Lcb (hocb) was amplified by polymerase chain reaction (PCR). We used L. casei 393 as an antigen delivery system to construct the recombinant strain pPG-2-hocb/L. casei 393. Then the recombinants were induced to express hocb. The immunoreactive band corresponding to hocb was observed by western-blotting, demonstrating the efficiency and specificity of hocb expression in recombinants. The measurements of optical density at 600 nm (OD600) after induction showed that expression of hocb can be used to convert L. casei cells into BGs. TEM showed that the cytomembrane and cell walls of hocb expressing cells were partially disrupted, accompanied by the loss of cellular contents, whereas control cells did not show any morphological changes. SEM showed that lysis pores were distributed in the middle or at the poles of the cells. To examine where the plasmid DNA was associated, we analyzed the L. casei ghosts loading SYBR Green I labeled pCI-EGFP by confocal microscopy. The result demonstrated that the DNA interacted with the inside rather than with the outside surface of the BGs. To further analyze where the DNA were loaded, we stained BGs with MitoTracker Green FM and the loaded plasmids were detected using EGFP-specific Cy-3-labeled probes. Z-scan sections through the BGs revealed that pCI-EGFP (red) was located within the BGs (green), but not on the outside. Flow cytometry and qPCR showed that the DNA was loaded onto BGs effectively and stably. Conclusions Our study constructed L. casei BGs by a novel method, which may be a promising technology for promoting the further application of DNA vaccine, providing experimental data to aid the development of other Gram-positive BGs.

// Gayeon Won 1 , Boram Kim 1 and John Hwa Lee 1 1 College of Veterinary Medicine, Chonbuk National University, Iksan Campus, Iksan, Republic of Korea Correspondence to: John Hwa Lee, email: johnhlee@jbnu.ac.kr Keywords: Salmonella Typhi, holin-endolysin system, bacterial ghosts, inactivated vaccine, typhoid fever Received: April 07, 2017 Accepted: May 04, 2017 Published: June 06, 2017 ABSTRACT Human typhoid fever caused by Salmonella Typhi still poses a severe global disease burden in developing countries despite the availability of commercial vaccines. In this study, we constructed a non-living S . Typhi Ty21a vaccine candidate by employing a lambda (λ) phage-derived holin-endolysin system to efficiently construct bacterial ghosts. The lysis plasmid pJHL464 harbors an R lysis cassette that is stringently regulated by dual promoters containing cI857/λPR and P araBAD /araC components. The plasmid was introduced into an asd gene-deleted S . Typhi Ty21a strain designated JOL1675. The in vitro expression of endolysin (~17.76 kDa) in the subsequent JOL1675 vaccine construct when grown under lysis inducible conditions was validated by immunoblotting. In scanning electron microscopy analysis, surface transmembrane tunnels and a collapsed body were visualized in the ghosts. Following 48 h of lysis, no viable JOL1675 cells remained, indicating that lysis of all cells was achieved. Subcutaneous immunizations of mice with the JOL1675 ghosts produced significantly increasing titers of serum IgG and vaginal wash secretory IgA antibodies against JOL1675 outer membrane proteins during the observational period. Further, serum collected at 6 weeks post-immunization of rabbits exhibited effective bactericidal activity against wild type S . Typhi in the presence of complement. These data showed that JOL1675 ghosts are highly immunogenic and elicit humoral and mucosal responses expected to correlate with protective immunity against S . typhi. Collectively, our findings support the conclusion that incorporating a λ phage holin-endolysin-mediated lysis construct into S . Typhi is an efficient strategy for developing a novel and safe non-living typhoid vaccine candidate.

Antibiotic resistance has become a major risk to community health over last few years because of antibiotics overuse around the globe and lack of new antibiotics development. Phages and their lytic enzymes are considered as an effective alternative of antibiotics to control drug resistant bacterial pathogens. Endolysins prove to be a promising class of antibacterials due to their specificity and less chances of resistance development in bacterial pathogens. Though large number of endolysins has been reported against gram positive bacteria, very few reported against gram negative bacteria due to the presence of outer membrane, which acts as physical barrier against endolysin attack to peptidoglycan. In the current study, we have expressed endolysin (RL_Lys) and holin fused at the N terminus of endolysin (RL_Hlys) from RL phage infecting multi drug resistant (MDR) Pseudomonas aeruginosa. Both endolysin variants were found active against wide range of MDR strains P. aeruginosa, Klebsella pneumonia, Salmonella Sp. and Methicillin Resistant Staphylococcus aureus (MRSA). Broth reduction assay showed that RL_Hlys is more active than RL_Lys due to presence of holin, which assist the endolysin access towards cell wall. The protein ligand docking and molecular dynamic simulation results showed that C- terminus region of endolysin play vital role in cell wall binding and even in the absence of holin, hydrolyze a broad range of gram negative bacterial pathogens. The significant activity of RL-Lys and RL_Hlys against a broad range of MDR gram negative and positive bacterial pathogens makes them good candidates for antibiotic alternatives.

Double-stranded DNA bacteriophages employ holin and endolysin functions to lyse host bacteria after virus multiplication. Holins oligomerize in the cytoplasmic membrane and trigger to form holes that cause cell death. For most systems these holes are also required for endolysin release to the cell wall, where it cleaves the peptidoglycan network. Orfs 25 and 26 of Bacillus subtilis phage SPP1 were predicted to encode the endolysin and holin functions, respectively. However, the product of the upstream orf 24.1 exhibits also holin features. We show that production of gp24.1 or gp26 in B. subtilis causes no major impact on cell growth, despite their ability to insert in the cytoplasmic membrane. Instant growth cessation and cell death is observed only upon co-production of the two holin-like proteins. Surprisingly, a constitutive promoter was identified within orf 24.1, which we propose to correspond to the previously described SPP1 early promoter PE5.

Bacteriophages (phages), or bacterial viruses, have recently received increasing attention, especially considering pan-drug-resistant bacteria, and studies on lytic bacteriophage proteins would help develop antibiotic candidates to treat these bacterial infections. We previously isolated and sequenced a Streptomyces avermitilis bacteriophage, phiSASD1. This study aimed to clone and express ORF40 and ORF19, previously predicted as endolysin (termed LytSD) and holin (termed HolSD), two crucial phage proteins involved in host lysis. The yield of LytSD was 17.2 mg per liter of culture, and the optimal lysis conditions were investigated. When applied exogenously, LytSD lysed 7/18 of the tested bacterial strains, including S. avermitilis, Bacillus subtilis, Staphylococcus aureus, Sarcina lutea, and Enterococcus faecalis. As regards HolSD, it resulted in growth inhibition of several tested strains and abrupt lysis of E. coli BL21 (DE3) pLysS; furthermore, it complemented the defective λ S allele of non-suppressing E. coli strains to produce phage plaques. Together, these results indicate the function of ORF40 and ORF19 of phage phiSASD1 and their potentials as novel antibiotics to inhibit or lyse pathogens.

Double-strand DNA bacteriophages employ the holin-endolysin dyad as core components of different strategies to lyse bacterial hosts. In the so-called canonical model the holin holes play an essential role in lysis as they provide a conduit for passage of the cytoplasm-accumulated endolysin to the cell wall (CW), where it degrades the peptidoglycan. It is considered that once synthesized canonical endolysins immediately acquire their fully active conformation, having thus the capacity to efficiently cleave the peptidoglycan if contact to the CW is allowed. We show here however that holin-mediated cell death may be required to fully sensitize cells to the lytic action of canonical endolysins, a role that is obviously masked by the key function of the holin in endolysin release. We demonstrate that in certain conditions Bacillus subtilis cells are capable of counteracting the activity of the phage SPP1 endolysin attacking the CW either from within or from without. This capacity is lost after holin action or in presence of agents that mimic its membrane-depolarizing role. We have observed a similar relationship between lytic activity and membrane proton motive force for a staphylococcal endolysin. The possible implications of these findings in the exploitation of endolysins as enzybiotics are discussed.

Bacterial cell lysis during bacteriophage infection is timed by perfect orchestration between components of the holin-endolysin cassette. In bacteria, progressively accumulating holin in the inner membrane, retained in its inactive form by antiholin, is triggered into active hole formation, resulting in the canonical host cell lysis. However, the molecular mechanism of regulation and physical basis of pore formation in the mycobacterial cell membrane by D29 mycobacteriophage holin, particularly in the nonexistence of a known antiholin, is poorly understood. In this study, we report, for the first time, the use of fluorescence resonance transfer measurements to demonstrate that the first transmembrane domain (TM1) of D29 holin undergoes a helix ↔ β-hairpin conformational interconversion. We validate that this structural malleability is mediated by a centrally positioned proline and is responsible for controlled TM1 self-association in membrana, in the presence of a proton gradient across the lipid membrane. We demonstrate that TM1 is sufficient for bacterial growth inhibition. The biological effect of D29 holin structural alteration is presented as a holin self-regulatory mechanism, and its implications are discussed in the context of holin function.

Yersinia enterocolitica is a pathogen that causes gastroenteritis in humans. Because of its low-temperature-dependent insecticidal activity, it can oscillate between invertebrates and mammals as host organisms. The insecticidal activity of strain W22703 is associated with a pathogenicity island of 19 kb (Tc-PAI Ye ), which carries regulators and genes encoding the toxin complex (Tc). The island also harbors four phage-related and highly conserved genes of unknown functions, which are polycistronically transcribed. Two open reading frames showed significant homologies to holins and endolysins and exhibited lytic activity in Escherichia coli cells upon overexpression. When a set of Yersinia strains was tested in an equivalent manner, highly diverse susceptibilities to lysis were observed, and some strains were resistant to lysis. If cell lysis occurred (as demonstrated by membrane staining), it was more pronounced when two accessory elements of the cassette coding for an i-spanin and an o-spanin were included in the overexpression construct. The pore-forming function of the putative holin, HolY, was demonstrated by complementation of the lysis defect of a phage λ S holin mutant. In experiments performed with membrane preparations, ElyY exhibited high specificity for W22703 peptidoglycan, with a cleavage activity resembling that of lysozyme. Although the functionality of the lysis cassette from Tc-PAI Ye was demonstrated in this study, its biological role remains to be elucidated.IMPORTANCE The knowledge of how pathogens survive in the environment is pivotal for our understanding of bacterial virulence. The insecticidal and nematocidal activity of Yersinia spp., by which the bacteria gain access to nutrients and thus improve their environmental fitness, is conferred by the toxin complex (Tc) encoded on a highly conserved pathogenicity island termed Tc-PAI Ye While the regulators and the toxin subunits of the island had been characterized in some detail, the role of phage-related genes within the island remained to be elucidated. Here, we demonstrate that this cassette encodes a holin, an endolysin, and two spanins that, at least upon overexpression, lyse Yersinia strains.

Programmed cell death in bacteria is generally triggered by membrane proteins with functions analogous to those of bacteriophage holins: they disrupt the membrane potential, whereas antiholins antagonize this process. The holin-like class of proteins is present in all three domains of life, but their functions can be different, depending on the species. Using a series of biochemical and genetic approaches, in a recent article in mBio , Charbonnier et al. (mBio 8:e00976-17, 2017, https://doi.org/10.1128/mBio.00976-17 ) demonstrate that the antiholin homologue in Bacillus subtilis transports pyruvate and is regulated in an unconventional way by its substrate molecule. Here, we discuss the connection between cell death and metabolism in various bacteria carrying genes encoding these holin-antiholin analogues and place the recent study by Charbonnier et al. in an evolutionary context.