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Lytic phages control the timepoint of host cell lysis by timing the holin-mediated release of cell wall-degrading endolysins. In phage T4, the antiholin RI inhibits the holin T, thereby preventing the early release of the T4 endolysin and lysis. The antiholin achieves lysis inhibition (LIN) in response to phage superinfections, thereby increasing the chance for lysis in an environment with a lower phage concentration. The holin T consists of a small N-terminal cytoplasmic domain, a transmembrane helix, and a periplasmic C-terminal domain. The antiholin is targeted to the periplasm by a cleavable signal peptide. Recently, the periplasmic soluble domains of the holin and the antiholin were found to form T2/RI2 tetramers in crystals. To investigate the functional relevance of this complex, we reconstituted LIN in a phage-free system, using only RI, T, and endolysin, and combined targeted mutagenesis with functional analyses. Inactivation of the RI signal peptide cleavage site did not abolish LIN, indicating that RI can function in a membrane-bound state, which argued against the tetramer. This led to analyses showing that only one of the two T/RI interfaces in the tetramer is physiologically relevant, which is also the only interaction site predicted by AlphaFold2. Some holin mutations at this interaction site prevented lysis, suggesting that the RI interaction likely acts by blocking the holin oligomerization required for hole formation. We conclude that LIN is mediated by a dimeric T/RI complex that, unlike the tetramer, can be easily formed when both partners are membrane-anchored. [ABSTRACT FROM AUTHOR]

Bacterial ghosts (BGs) are described as bacterial cell envelopes that retain their structure but lack cytoplasmic contents. The study of BGs spans multiple disciplinary domains, and the development of BG production techniques to obtain ample and stable BG samples holds significant implications for probing the biological characteristics of BGs, devising novel disease treatment strategies, and leveraging their industrial applications. Numerous products encoded within bacteriophage (phage) genomes possess the capability to lyse bacteria, thereby inducing BG formation primarily via disruption of bacterial cell wall integrity. This review comprehensively surveys the utilization of phage-encoded proteins in BG production techniques, encompassing methodologies such as phage E protein-mediated lysis, perforin protein-induced lysis, and strategies combining E protein with holin-endolysin systems. Additionally, discussions and summaries are provided on the current applications, challenges, and modification strategies associated with different techniques. Through a focused exploration of BG production techniques, with an emphasis on precise manipulation of BG formation using phage-encoded protein technologies, this study aims to furnish robust tools and methodologies for delving into the mechanisms underlying BG formation, as well as for the development of novel therapeutic strategies and applications based on BGs. [ABSTRACT FROM AUTHOR]

The analysis of transcriptional activity of the bacteriophage T5 hol/endo operon conducted in the paper revealed a strong constitutive promoter recognized by E. coli RNA polymerase and a transcription initiation point of the operon. It was also shown that the only translational start codon for holin was a non-canonical TTG. Translation initiation regions (TIRs) of both genes of the operon (hol and endo) were further analyzed using chimeric constructs, in which parts of the hol/endo regulatory regions were fused with the gene of a reporter protein (EGFP). It was found that TIR of hol was 20 times less effective than that of endo. As it turned out, the level of EGFP production was influenced by the composition of the constructs and the type of the hol start codon. Apparently, the translational suppression of holin's accumulation and posttranslational activation of endolysin by Ca2+ are the main factors ensuring the proper timing of the host cell lysis by bacteriophage T5. The approach based on the use of chimeric constructs proposed in the paper can be recommended for studying other native or artificial operons of any complexity: analyzing the impacts of separate DNA regions, as well as their coupled effect, on the processes of transcription and translation of recombinant protein(s). [ABSTRACT FROM AUTHOR]

Lytic phages control the timepoint of host cell lysis by timing the holin-mediated release of cell wall-degrading endolysins. In phage T4, the antiholin RI inhibits the holin T, thereby preventing the early release of the T4 endolysin and lysis. The antiholin achieves lysis inhibition (LIN) in response to phage superinfections, thereby increasing the chance for lysis in an environment with a lower phage concentration. The holin T consists of a small N-terminal cytoplasmic domain, a transmembrane helix, and a periplasmic C-terminal domain. The antiholin is targeted to the periplasm by a cleavable signal peptide. Recently, the periplasmic soluble domains of the holin and the antiholin were found to form T2/RI2 tetramers in crystals. To investigate the functional relevance of this complex, we reconstituted LIN in a phage-free system, using only RI, T, and endolysin, and combined targeted mutagenesis with functional analyses. Inactivation of the RI signal peptide cleavage site did not abolish LIN, indicating that RI can function in a membrane-bound state, which argued against the tetramer. This led to analyses showing that only one of the two T/RI interfaces in the tetramer is physiologically relevant, which is also the only interaction site predicted by AlphaFold2. Some holin mutations at this interaction site prevented lysis, suggesting that the RI interaction likely acts by blocking the holin oligomerization required for hole formation. We conclude that LIN is mediated by a dimeric T/RI complex that, unlike the tetramer, can be easily formed when both partners are membrane-anchored.

Ralstonia solanacearum, a pathogen causing widespread bacterial wilt disease in numerous crops, currently lacks an optimal control agent. Given the limitations of traditional chemical control methods, including the risk of engendering drug-resistant strains and environmental harm, there is a dire need for sustainable alternatives. One alternative is lysin proteins that selectively lyse bacteria without contributing to resistance development. This work explored the biocontrol potential of the LysP2110-HolP2110 system of Ralstonia solanacearum phage P2110. Bioinformatics analyses pinpointed this system as the primary phage-mediated host cell lysis mechanism. Our data suggest that LysP2110, a member of the Muraidase superfamily, requires HolP2110 for efficient bacterial lysis, presumably via translocation across the bacterial membrane. LysP2110 also exhibits broad-spectrum antibacterial activity in the presence of the outer membrane permeabilizer EDTA. Additionally, we identified HolP2110 as a distinct holin structure unique to the Ralstonia phages, underscoring its crucial role in controlling bacterial lysis through its effect on bacterial ATP levels. These findings provide valuable insights into the function of the LysP2110-HolP2110 lysis system and establish LysP2110 as a promising antimicrobial agent for biocontrol applications. This study underpins the potential of these findings in developing effective and environment-friendly biocontrol strategies against bacterial wilt and other crop diseases. [ABSTRACT FROM AUTHOR]

The insecticidal toxin complex (Tc) proteins are produced by several insect-associated bacteria, including Yersinia enterocolitica strain W22703, which oscillates between two distinct pathogenicity phases in invertebrates and humans. The mechanism by which this high-molecular-weight toxin is released into the extracellular surrounding, however, has not been deciphered. In this study, we investigated the regulation and functionality of a phage-related holin/endolysin (HE) cassette located within the insecticidal pathogenicity island Tc-PAIYe of W22703. Using the Galleria mellonella infection model and luciferase reporter fusions, we revealed that quorum sensing contributes to the insecticidal activity of W22703 upon influencing the transcription of tcaR2, which encodes an activator of the tc and HE genes. In contrast, a lack of the Yersinia modulator, YmoA, stimulated HE gene transcription, and mutant W22703 ΔymoA exhibited a stronger toxicity toward insect larvae than did W22703. A luciferase reporter fusion demonstrated transcriptional activation of the HE cassette in vivo, and a significantly larger extracellular amount of subunit TcaA was found in W22703 ΔymoA relative to its ΔHE mutant. Using competitive growth assays, we demonstrated that at least in vitro, the TcaA release upon HE activity is not mediated by cell lysis of a significant part of the population. Oral infection of Caenorhabditis elegans with a HE deletion mutant attenuated the nematocidal activity of the wild type, similar to the case with a mutant lacking a Tc subunit. We conclude that the dual holin/endolysin cassette of yersiniae is a novel example of a phage-related function adapted for the release of a bacterial toxin. IMPORTANCE Members of the genus Yersinia cause gastroenteritis in humans but also exhibit toxicity toward invertebrates. A virulence factor required for this environmental life cycle stage is the multisubunit toxin complex (Tc), which is distinct from the insecticidal toxin of Bacillus thuringiensis and has the potential to be used in pest control. The mechanism by which this high-molecular-weight Tc is secreted from bacterial cells has not been uncovered. Here, we show that a highly conserved phage-related holin/endolysin pair, which is encoded by the genes holY and elyY located between the Tc subunit genes, is essential for the insecticidal activity of Y. enterocolitica and that its activation increases the amount of Tc subunits in the supernatant. Thus, the dual holY-elyY cassette of Y. enterocolitica constitutes a new example for a type 10 secretion system to release bacterial toxins. [ABSTRACT FROM AUTHOR]

Abstract Background The holin-endolysin lysis system plays an essential role in the phage life cycle. Endolysins are promising alternatives to antibiotics, and have been successfully used against Gram-positive bacteria. However, a few endolysins can externally lyse Gram-negative bacteria, due to the inaccessible peptidoglycan layer covered by the envelope. Results This study investigated the lysis system of a new Siphoviridae bacteriophage vB_Sal-S-S10 (S10), which, that was isolated from broiler farms, was found to be able to infect 51.4% (37/72) of tested S. enteritidis strains. Phage S10 genome had a classic holin-endolysin lysis system, except that one holin and one endolysin gene were functionally annotated. The orf 22 adjacent to the lysis cassette was identified as a new endolysin gene. Antibacterial activity assays showed that holin had an intracellular penetrating activity against S. enteritidis 35; both endolysins acted on the cell envelope of S. enteritidis 35 and showed a natural extracellular antibacterial activity, leading to a ~ 1 log titer decrease in 30 min. Protein characterization of lysin1 and lysin2 revealed that the majority of the N-terminus and the C-terminus were hydrophobic amino acids or positively charged. Conclusion In this study, a new Salmonella phage vB_Sal-S-S10 (S10) was characterized and showed an ideal development prospect. Phage S10 has a classic holin-endolysin lysis system, carrying an overlapping holin-lysin gene and a novel lysin gene. Both endolysins coded by lysin genes could externally lyse S. enteritidis. The natural extracellular antibacterial character of endolysins would provide necessary information for the development of engineering endolysin as the antibiotic alternative against the infection with multidrug-resistant gram-negative bacteria.

In this study, we isolated a lytic Pseudomonas aeruginosa phage (vB_PaeP_ASP23) from the sewage of a mink farm, characterized its complete genome and analyzed the function of its putative lysin and holin. Morphological characterization and genome annotation showed that phage ASP23 belonged to the Krylovirinae family genus Phikmvvirus, and it had a latent period of 10 min and a burst size of 140 pfu/infected cell. In minks challenged with P. aeruginosa, phage ASP23 significantly reduced bacterial counts in the liver, lung, and blood. The whole-genome sequencing showed that its genome was a 42,735-bp linear and double-stranded DNA (dsDNA), with a G + C content of 62.15%. Its genome contained 54 predicted open reading frames (ORFs), 25 of which had known functions. The lysin of phage ASP23 (LysASP), in combination with EDTA, showed high lytic activity against P. aeruginosa L64. The holin of phage ASP23 was synthesized by M13 phage display technology, to produce recombinant phages (HolASP). Though HolASP exhibited a narrow lytic spectrum, it was effective against Staphylococcus aureus and Bacillus subtilis. However, these two bacteria were insensitive to LysASP. The findings highlight the potential of phage ASP23 to be used in the development of new antibacterial agents. [ABSTRACT FROM AUTHOR]

Bacterial ghosts (BGs) are nonviable empty bacterial cell envelopes with intact cellular morphology and native surface structure. BGs made from pathogenic bacteria are used for biomedical and pharmaceutical applications. However, incomplete pathogenic cell inactivation during BG preparation raises safety concerns that could limit the intended use. Therefore, safer bacterial cell types are needed for BG production. Here, we produced BGs from the food-grade Gram-positive bacterium Lactobacillus plantarum TBRC 2–4 by conditional expression of a prophage-encoded holin (LpHo). LpHo expression was regulated using the pheromone-inducible pSIP system and LpHo was localized to the cell membrane. Upon LpHo induction, a significant growth retardation and a drastic decrease in cell viability were observed. LpHo-induced cells also showed membrane pores by scanning electron microscopy, membrane depolarization by flow cytometry, and release of nucleic acid contents in the cell culture supernatant, consistent with the role of LpHo as a pore-forming protein and L. plantarum ghost formation. The holin-induced L. plantarum BG platform could be developed as a safer alternative vehicle for the delivery of biomolecules. [ABSTRACT FROM AUTHOR]

Background: The holin-endolysin lysis system plays an essential role in the phage life cycle. Endolysins are promising alternatives to antibiotics, and have been successfully used against Gram-positive bacteria. However, a few endolysins can externally lyse Gram-negative bacteria, due to the inaccessible peptidoglycan layer covered by the envelope. Results: This study investigated the lysis system of a new Siphoviridae bacteriophage vB_Sal-S-S10 (S10), which, that was isolated from broiler farms, was found to be able to infect 51.4% (37/72) of tested S. enteritidis strains. Phage S10 genome had a classic holin-endolysin lysis system, except that one holin and one endolysin gene were functionally annotated. The orf 22 adjacent to the lysis cassette was identified as a new endolysin gene. Antibacterial activity assays showed that holin had an intracellular penetrating activity against S. enteritidis 35; both endolysins acted on the cell envelope of S. enteritidis 35 and showed a natural extracellular antibacterial activity, leading to a ~ 1 log titer decrease in 30 min. Protein characterization of lysin1 and lysin2 revealed that the majority of the N-terminus and the C-terminus were hydrophobic amino acids or positively charged. Conclusion: In this study, a new Salmonella phage vB_Sal-S-S10 (S10) was characterized and showed an ideal development prospect. Phage S10 has a classic holin-endolysin lysis system, carrying an overlapping holin-lysin gene and a novel lysin gene. Both endolysins coded by lysin genes could externally lyse S. enteritidis. The natural extracellular antibacterial character of endolysins would provide necessary information for the development of engineering endolysin as the antibiotic alternative against the infection with multidrug-resistant gram-negative bacteria. [ABSTRACT FROM AUTHOR]

A simple generic method for enhancing extracellular protein yields in engineered bacteria is still lacking. Here, we demonstrated that phage‐encoded holin can be used to export proteins to the extracellular medium in both Gram‐negative Escherichia coli and ‐positive Lactococcus lactis. When a putative holin gene LLNZ_RS10380 annotated in the genome of L. lactis NZ9000 (hol380) was recombinantly expressed in E. coli BL21(DE3), the Hol380 oligomerized up to hexamer in the cytoplasmic membrane, yielding membrane pore to allow the passage of cytosolic β‐galatosidase (116 kDa), whose extracellular production reached 54.59 U/μl, accounting for 76.37% of the total activity. However, the overexpressed Hol380 could not release cytosolic proteins across the membrane in L. lactis NZ9000, but increased the secretory production of staphylococcal nuclease to 2.55‐fold and fimbrial adhesin FaeG to 2.40‐fold compared with those guided by signal peptide Usp45 alone. By using a combination of proteomics and transcriptional level analysis, we found that overexpression of the Hol380 raised the accumulation of Ffh and YidC involved in the signal recognition particle pathway in L. lactis, suggesting an alternative road participating in protein secretion. This study proposed a new approach by expressing holin in bacterial cell factories to export target proteins of economic or medical interest. [ABSTRACT FROM AUTHOR]

Ralstonia solanacearum, a pathogen causing widespread bacterial wilt disease in numerous crops, currently lacks an optimal control agent. Given the limitations of traditional chemical control methods, including the risk of engendering drug-resistant strains and environmental harm, there is a dire need for sustainable alternatives. One alternative is lysin proteins that selectively lyse bacteria without contributing to resistance development. This work explored the biocontrol potential of the LysP2110-HolP2110 system of Ralstonia solanacearum phage P2110. Bioinformatics analyses pinpointed this system as the primary phage-mediated host cell lysis mechanism. Our data suggest that LysP2110, a member of the Muraidase superfamily, requires HolP2110 for efficient bacterial lysis, presumably via translocation across the bacterial membrane. LysP2110 also exhibits broad-spectrum antibacterial activity in the presence of the outer membrane permeabilizer EDTA. Additionally, we identified HolP2110 as a distinct holin structure unique to the Ralstonia phages, underscoring its crucial role in controlling bacterial lysis through its effect on bacterial ATP levels. These findings provide valuable insights into the function of the LysP2110-HolP2110 lysis system and establish LysP2110 as a promising antimicrobial agent for biocontrol applications. This study underpins the potential of these findings in developing effective and environment-friendly biocontrol strategies against bacterial wilt and other crop diseases.

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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. IMPORTANCE The phage life cycle ends with the host cell lysis, thereby releasing new virions into the environment for the next round of bacterial infection. Nowadays, there is renewed interest in phages as biocontrol agents, primarily due to their ability to cause bacterial death through lysis. While endolysins, which mediate peptidoglycan degradation, have been fairly well described, the pore-forming proteins, referred to as holins, have been extensively characterized in only a few model phages, mainly infecting Gram-negative bacteria. In this work, we characterized the holins encoded by a siphovirus and two myoviruses targeting members of the Gram-positive Bacillus cereus group, which comprises closely related species, including the well-known Bacillus anthracis, B. cereus sensu stricto, and Bacillus thuringiensis. Overall, this paper provides the first experimental characterization of holins encoded by B. cereus phages and reveals versatile lysis mechanisms used by these phages. [ABSTRACT FROM AUTHOR]

Bacteriophages (phages) infecting specifically target bacteria utilize a unique lysis module known as the holin-endolysin cassette to release progeny. Studies on the phage lytic proteins could contribute to the development of alternatives to antibiotics. Here, we predicted and identified the holin protein of rV5-like phage ECP26 for increasing lytic activity of the phage endolysin. In silico analysis revealed that open reading frame 151 (ORF151) of ECP26 contained two transmembrane domains. Co-expression of endolysin with ORF151 resulted in the cell lysis of Escherichia coli, suggesting that ORF151 protein functioned as the holin that disrupted the cytoplasmic membrane. The putative holin showed a high amino acid homology by more than 80% to the predicted holins of rV5-like phages. Therefore, the holin protein would be helpful for developing efficient lysis strategies with endolysin against gram-negative E. coli. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance represents a global health emergency, necessitating the introduction of novel antimicrobial agents. In the present study, lysozyme and holin from Shigella flexneri 1.1868 phage SGF2, named LysSGF2 and HolSGF2, respectively, were cloned, expressed, and characterized. LysSGF2 and HolSGF2 showed lytic activities against S. flexneri 1.1868 cells at 4–55 °C and pH 3.1–10.3. LysSGF2 exhibited antimicrobial activity against five gram-negative and two gram-positive bacteria. HolSGF2 showed antimicrobial activity against four gram-negative and one gram-positive species. The antibacterial activities of LysSGF2 and HolSGF2 were determined in liquid beverages, including bottled water and milk. The relative lytic activity of LysSGF2 combined with HolSGF2 against the tested bacteria was approximately 46–77 % in water. Furthermore, the combination markedly decreased the viable counts of tested bacteria by approximately 3–5 log CFU/mL. LysSGF2 and HolSGF2 could efficiently remove biofilms on polystyrene, glass, and stainless-steel. The efficacy of the LysSGF2 and HolSGF2 combination against the tested bacteria on polystyrene was 58–71 %. Combination treatment effectively killed biofilm cells formed on stainless-steel and glass by 1–4 log CFU/mL. ese results indicate that LysSGF2 and HolSGF2 can successfully control both the planktonic and biofilm cells of common pathogenic bacteria, suggesting that the combined or single use of LysSGF2 and HolSGF2 may be of great value in food processing. • Lysozyme and holin(removed signal peptide and a transmembrane region) from Shigella flexneri 1.1868 phage SGF2 were characterized. • LysSGF2 and HolSGF2 lytic activity was effective in a wide temperature and pH range. • Both showed antimicrobial activity against gram-positive and -negative species. • Combined holin and lysozyme treatment efficiently killed bacteria in liquids. • Combination treatment killed bacterial biofilms formed on polystyrene plastic, steel and glass. [ABSTRACT FROM AUTHOR]

ABSTRACT Optimality models have a checkered history in evolutionary biology. While optimality models have been successful in providing valuable insight into the evolution of a wide variety of biological traits, a common objection is that optimality models are overly simplistic and ignore organismal genetics. We revisit evolutionary optimization in the context of a major bacteriophage life history trait, lysis time. Lysis time refers to the period spanning phage infection of a host cell and its lysis, whereupon phage progenies are released. Lysis time, therefore, directly determines phage fecundity assuming progeny assembly does not exhaust host resources prior to lysis. Noting that previous tests of lysis time optimality rely on batch culture, we implemented a quasi-continuous culture system to observe productivity of a panel of isogenic phage λ genotypes differing in lysis time. We report that under our experimental conditions, λ phage productivity is maximized around optimal lysis times ranging from 60 to 100 min, and λ wildtype strain falls within this range. It would appear that natural selection on phage λ lysis time uncovered a set of genetic solutions that optimized progeny production in its ecological milieu relative to alternative genotypes. We discuss this finding in light of recent results that lysis time variation is also minimized in the strains with lysis times closer to the λ wild-type strain. IMPORTANCE Optimality theory presents the idea that natural selection acts on organismal traits to produce genotypes that maximize organismal survival and reproduction. As such, optimality theory is a valuable tool in guiding our understanding of the genetic constraints and tradeoffs organisms experience as their genotypes are selected to produce optimal solutions to biological problems. However, optimality theory is often critiqued as being overly simplistic and ignoring the roles of chance and history in the evolution of organismal traits. We show here that the wild-type genotype of a popular laboratory model organism, the bacteriophage λ, produces a phenotype for a major life history trait, lysis time, that maximizes the reproductive success of bearers of that genotype relative to other possible genotypes. This result demonstrates, as is rarely shown experimentally, that natural selection can achieve optimal solutions to ecological challenges.

A cyanophage PaV-LD, previously isolated from harmful filamentous cyanobacterium Planktothrix agardhii , was sequenced, and co-expression of its two ORFs in tandem, ORF123 and ORF124, inhibited growth on the model cyanobacterium Synechocystis sp. PCC6803 cells. However, the mechanism of action of ORF123 and ORF124 alone remains to be elucidated. In this study, we aimed to study the individual function of ORF123 or ORF124 from PaV-LD. Our data showed that the ORF123 encoded an endopeptidase, which harbored an M23 family peptidase domain and a transmembrane region. The expression of the endopeptidase in Escherichia coli alone revealed that the protein exhibited remarkable bacteriostatic activity, as evidenced by observation of growth inhibition, membrane damage, and leakage of the intracellular enzyme. Similarly, the holin, a membrane-associated protein encoded by the ORF124, showed weak bacteriostatic activity on E. coli. Moreover, deletion mutations indicated that the transmembrane domains of endopeptidase and holin were indispensable for their bacteriostatic activity. Meanwhile, the bacteriostatic functions of endopeptidase and holin on cyanobacteria cells were confirmed by expressing them in the cyanobacterium Synechocystis sp. PCC6803. Collectively, our study revealed the individual role of endopeptidase or holin and their synergistic bacteriolytic effect, which would contribute to a better understanding of the lytic mechanism of cyanophage PaV-LD. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In the current study, we evaluated the impacts of organic and chemical fertilizers as well as the foliar application of growth regulators on quantitative traits and seed oil content of safflower grown in northwestern Iran. The experiment was done as a split-plot (3 × 5), and the main plot was assigned to different fertilizers, including organic fertilizer (FYM: farmyard manure 20 t ha-1), full chemical fertilizer (FCF) and no fertilization "control" (NF). The sub-plots were allocated to foliar spray treatments, which included control (no-spray application; S0), choline (S1), chitosan (S2) and salicylic acid (S3). The results showed that the application of fertilizers significantly affected the oil and protein content of the seed. However, the effect of FYM was more prominent than FCF on oil qualitative characteristics. A significant positive correlation was observed between oil content, protein content, and some fatty acid composition such as oleic acid, arachidonic acid, stearic acid, and palmitic acid. The highest values of the previously mentioned traits were obtained using FYMS1, FCFS1 and FYMS3. This trend was also clearly obvious in the content of linoleic acid as the main fatty acid in safflower oil. Among the foliar spraying treatments, the most improving effect was obtained with the use of choline. In conclusion, improving soil conditions through the application of FYM and appropriate amounts of chemical fertilizers is one of the most important agronomic management measures to improve the oil quality of safflower seeds. [ABSTRACT FROM AUTHOR]

A cyanophage PaV-LD, previously isolated from harmful filamentous cyanobacterium Planktothrix agardhii, was sequenced, and co-expression of its two ORFs in tandem, ORF123 and ORF124, inhibited growth on the model cyanobacterium Synechocystis sp. PCC6803 cells. However, the mechanism of action of ORF123 and ORF124 alone remains to be elucidated. In this study, we aimed to study the individual function of ORF123 or ORF124 from PaV-LD. Our data showed that the ORF123 encoded an endopeptidase, which harbored an M23 family peptidase domain and a transmembrane region. The expression of the endopeptidase in Escherichia coli alone revealed that the protein exhibited remarkable bacteriostatic activity, as evidenced by observation of growth inhibition, membrane damage, and leakage of the intracellular enzyme. Similarly, the holin, a membrane-associated protein encoded by the ORF124, showed weak bacteriostatic activity on E. coli. Moreover, deletion mutations indicated that the transmembrane domains of endopeptidase and holin were indispensable for their bacteriostatic activity. Meanwhile, the bacteriostatic functions of endopeptidase and holin on cyanobacteria cells were confirmed by expressing them in the cyanobacterium Synechocystis sp. PCC6803. Collectively, our study revealed the individual role of endopeptidase or holin and their synergistic bacteriolytic effect, which would contribute to a better understanding of the lytic mechanism of cyanophage PaV-LD.

ABSTRACT The Staphylococcus aureus cidABC and lrgAB operons encode members of a well-conserved family of proteins thought to be involved in programmed cell death (PCD). Based on the structural similarities that CidA and LrgA share with bacteriophage holins, we have hypothesized that these proteins function by forming pores within the cytoplasmic membrane. To test this, we utilized a “lysis cassette” system that demonstrated the abilities of the cidA and lrgA genes to support bacteriophage endolysin-induced cell lysis. Typical of holins, CidA- and LrgA-induced lysis was dependent on the coexpression of endolysin, consistent with the proposed holin-like functions of these proteins. In addition, the CidA and LrgA proteins were shown to localize to the surface of membrane vesicles and cause leakage of small molecules, providing direct evidence of their hole-forming potential. Consistent with recent reports demonstrating a role for the lrgAB homologues in other bacterial and plant species in the transport of by-products of carbohydrate metabolism, we also show that lrgAB is important for S. aureus to utilize pyruvate during microaerobic and anaerobic growth, by promoting the uptake of pyruvate under these conditions. Combined, these data reveal that the CidA and LrgA membrane proteins possess holin-like properties that play an important role in the transport of small by-products of carbohydrate metabolism. IMPORTANCE The Staphylococcus aureus cidABC and lrgAB operons represent the founding members of a large, highly conserved family of genes that span multiple kingdoms of life. Despite the fact that they have been shown to be involved in bacterial PCD, very little is known about the molecular/biochemical functions of the proteins they encode. The results presented in this study reveal that the cidA and lrgA genes encode proteins with bacteriophage holin-like functions, consistent with their roles in cell death. However, these studies also demonstrate that these operons are involved in the transport of small metabolic by-products of carbohydrate metabolism, suggesting an intriguing link between these two seemingly disparate processes.

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. [ABSTRACT FROM AUTHOR]

Bacteriophage T7 gene 17.5 coding for the only known holin is one of the components of its lysis system, but the holin activity in T7 is more complex than a single gene, and evidence points to the existence of additional T7 genes with holin activity. In this study, a T7 phage with a gene 17.5 deletion (T7-△holin) was rescued and its biological characteristics and effect on cell lysis were determined. Furthermore, the genomic evolution of mutant phage T7-△holin during serial passage was assessed by whole-genome sequencing analysis. It was observed that deletion of gene 17.5 from phage T7 delays lysis time and enlarges the phage burst size; however, this biological characteristic recovered to normal lysis levels during serial passage. Scanning electron microscopy showed that the two opposite ends of E. coli BL21 cells swell post-T7-△holin infection rather than drilling holes on cell membrane when compared with T7 wild-type infection. No visible progeny phage particle accumulation was observed inside the E. coli BL21 cells by transmission electron microscopy. Following serial passage of T7-△holin from the 1st to 20th generations, the mRNA levels of gene 3.5 and gene 19.5 were upregulated and several mutation sites were discovered, especially two missense mutations in gene 19.5, which indicate a potential contribution to the evolution of the T7-△holin. Although the burst size of T7-△holin increased, high titer cultivation of T7-△holin was not achieved by optimizing the culture process. Accordingly, these results suggest that gene 19.5 is a potential lysis-related component that needs to be studied further and that the T7-△holin strain with its gene 17.5 deletion is not adequate to establish the high-titer phage cultivation process.

Bacteriophage T7 gene 17.5 coding for the only known holin is one of the components of its lysis system, but the holin activity in T7 is more complex than a single gene, and evidence points to the existence of additional T7 genes with holin activity. In this study, a T7 phage with a gene 17.5 deletion (T7-△holin) was rescued and its biological characteristics and effect on cell lysis were determined. Furthermore, the genomic evolution of mutant phage T7-△holin during serial passage was assessed by whole-genome sequencing analysis. It was observed that deletion of gene 17.5 from phage T7 delays lysis time and enlarges the phage burst size; however, this biological characteristic recovered to normal lysis levels during serial passage. Scanning electron microscopy showed that the two opposite ends of E. coli BL21 cells swell post-T7-△holin infection rather than drilling holes on cell membrane when compared with T7 wild-type infection. No visible progeny phage particle accumulation was observed inside the E. coli BL21 cells by transmission electron microscopy. Following serial passage of T7-△holin from the 1st to 20th generations, the mRNA levels of gene 3.5 and gene 19.5 were upregulated and several mutation sites were discovered, especially two missense mutations in gene 19.5 , which indicate a potential contribution to the evolution of the T7-△holin. Although the burst size of T7-△holin increased, high titer cultivation of T7-△holin was not achieved by optimizing the culture process. Accordingly, these results suggest that gene 19.5 is a potential lysis-related component that needs to be studied further and that the T7-△holin strain with its gene 17.5 deletion is not adequate to establish the high-titer phage cultivation process. [ABSTRACT FROM AUTHOR]

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 Cterminal 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. [ABSTRACT FROM AUTHOR]

Gram‐negative bacteria have evolved numerous pathways to secrete proteins across their complex cell envelopes. Here, we describe a protein secretion system that 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 involved 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). [ABSTRACT FROM AUTHOR]

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.

Phages utilize lysis systems to allow the release of newly assembled viral particles that kill the bacterial host. This is also the case for phage AP1, which infects the rice pathogen Acidovorax oryzae. However, how lysis occurs on a molecular level is currently unknown. We performed in silico bioinformatics analyses, which indicated that the lysis cassette contains a holin (HolAP) and endolysin (LysAP), which are encoded by two adjacent genes. Recombinant expression of LysAP caused Escherichia coli lysis, while HolAP arrested growth. Co-expression of both proteins resulted in enhanced lysis activity compared to the individual proteins alone. Interestingly, LysAP contains a C-terminal region transmembrane domain, which is different from most known endolysins where a N-terminal hydrophobic region is found, with the potential to insert into the membrane. We show that the C-terminal transmembrane domain is crucial for protein localization and bacterial lysis in phage AP1. Our study characterizes the new phage lysis cassette and the mechanism to induce cell disruption, giving new insight in the understanding of phage life cycles.

Abstract Background Biocontrol of bacterial pathogens by bacteriophages (phages) represents a promising strategy. Vibrio alginolyticus, a gram-negative bacterium, is a notorious pathogen responsible for the loss of economically important farmed marine animals. To date, few V. alginolyticus phages have been successfully isolated, and only three complete genome sequences of them have been released. The limited available phage resources and poor genomic data hamper research on V. alginolyticus phages and their applications for the biocontrol of V. alginolyticus. Results We isolated a phage, Vp670, against the V. alginolyticus strain E06333 and obtained its full genomic sequence. It contains 43,121 nucleotides with a GC content of 43.4%, and codes for 49 predicted open reading frames. Observation by electron microscope combined with phylogenetic analysis of DNA polymerase indicates that Vp670 belongs to the subfamily Autographivirinae in the family Podoviridae. orf3 (designated holA) and orf8 (designated cwlQ) are predicted to encode a holin (HolA) and an endolysin (CwlQ), respectively. Expression of holA alone or coexpression of holA and cwlQ from within arrested the growth of Escherichia coli and V. alginolyticus while the expression of cwlQ alone had no effect on the growth of them. Further observation by transmission electron microscopy revealed that the expression of holA vanished the outer membrane and caused the release of cellular contents of V. alginolyticus and the coexpression of holA and cwlQ directly burst the cells and caused a more drastic release of cellular contents. Expression of cwlQ alone in V. alginolyticus did not cause cytomorphological changes. Conclusions Phage Vp670 is a V. alginolyticus phage belonging to the family of Podoviridae. The genome of Vp670 contains a two-component lysis module, which is comprised of holA and cwlQ. holA is predicted to encode for the holin protein, HolA, and cwlQ is predicted to encode for the endolysin protein, CwlQ. Both holA and cwlQ likely play important roles during the release of phage progeny.

Optimal phage propagation depends on the regulation of the lysis of the infected host cell. In T4 phage infection, lysis occurs when the holin protein (T) forms lesions in the host membrane. However, the lethal function of T can be blocked by an antiholin (RI) during lysis inhibition (LIN). LIN sets if the infected cell undergoes superinfection, then the lysis is delayed until host/phage ratio becomes more favorable for the release of progeny. It has been thought that a signal derived from the superinfection is required to activate RI. Here we report structures that suggest a radically different model in which RI binds to T irrespective of superinfection, causing it to accumulate in a membrane as heterotetrameric 2RI–2T complex. Moreover, we show the complex binds non-specifically to DNA, suggesting that the gDNA from the superinfecting phage serves as the LIN signal and that stabilization of the complex by DNA binding is what defines LIN. Finally, we show that soluble domain of free RI crystallizes in a domain-swapped homotetramer, which likely works as a sink for RI molecules released from the RI–T complex to ensure efficient lysis. These results constitute the first structural basis and a new model not only for the historic LIN phenomenon but also for the temporal regulation of phage lysis in general. Unlabelled Image • Lysis control in T4 phage infection and in particular the phenomenon of lysis inhibition (LIN), allowing the phage to delay host cell lysis in response to superinfection, captivated scientists' attention for over seven decades. However, the mechanism of lytic function regulation and the molecular basis of the superinfection signal remained obscure. • We present the crystal structure of the complex between periplasmic domains of the T4 holin T and its inhibitor antiholin RI, identifying holin as a GAF domain protein and providing molecular details of the holin inhibition. The structure of free RI shows a unique fold, capable of undergoing a large conformational change and domain swapping, which facilitates the release of the holin from the complex upon lysis triggering. • Structural information on the holin–antiholin complex led us to re-examine the existing model for LIN and propose that the superinfection acts by stabilizing the complex, pointing to genomic DNA injected into the periplasm by secondary phage as a likely stabilizing ligand. We were able to demonstrate binding of DNA to the RI–T complex by two independent methods, thus establishing DNA as a superinfection signal. • Based on the structural and biochemical evidence, we propose a novel model for the lysis pathway and regulation in T4 phages, where holin accumulates in the membrane in the form of inhibited by antiholin complexes. Lysis triggers at the time when holins titrate out the antiholins. Free holin rafts disrupt transmembrane potential, driving antiholin out of the bilayer, which leads to holin–antiholin complex dissociation, liberating all accumulated holins to polymerize into a hole. Secondary phage DNA, trapped and sheared into fragments in the periplasm, binds to holin–antiholin complexes transiently stabilizing the inhibited form of holin and delaying the lysis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Enterococcus faecalis , a conditional pathogenic bacterium, is prevalent in the intestinal, oral, and reproductive tracts of humans and animals, causing a variety of infectious diseases. E. faecalis is the main species detected in secondary persistent infection from root canal therapy failure. Due to the abuse of antibacterial agents, E. faecalis has evolved its resistant ability. Therefore, it is difficult to treat clinical diseases infected by E. faecalis. Exploring new alternative drugs for treating E. faecalis infection is urgent. We cloned and expressed the gene of phage holin, purified the recombinant protein, and analyzed the antibacterial activity, lysis profile, and ability to remove bacterial biofilm. It showed that the crude enzyme of phage holin pEF191 exhibited superior bacterial inhibiting activity and a broader lysis host range compared to the parent phage PEf771. In addition, pEF191 demonstrated high efficacy in eliminating E. faecalis biofilm. The therapeutic results of the Sprague-Dawley (SD) rats model infected showed that pEf191 did not affect SD rats, indicating that pEF191 provided greater protection against E. faecalis infection in SD rats. Based on the 16 S rDNA data of SD rats intestinal microorganism population, holin pEF191 exhibited no impact on the diversity of intestinal microorganisms at the phylum and genus levels and improved the relative abundance of favorable bacteria. Thus, pEF191 may serve as a promising alternative to antibiotics in the management of E. faecalis infection. • A new holin gene from phage, infecting Enterococcus faecalis YN771, was cloned and expressed. • The crude enzyme of phage holin pEF191 exhibited superior bacterial inhibiting activity. • The pEF191 provided greater protection against E. faecalis infection in SD rats. [ABSTRACT FROM AUTHOR]

[Display omitted] • Bioluminescent reporter phage was developed for Cronobacter sakazakii detection. • Reporter phage can specifically detect live C. sakazakii cells. • Truncation of lytic phage's holin allows a more stable and intensified light signal. • Reporter phage detects 2 CFU/ml of C. sakazakii in applied food models within 7 h. As outbreaks of foodborne illness caused by the opportunistic pathogen Cronobacter sakazakii (Cs) continue to occur, particularly in infants consuming powdered infant formula (PIF), the need for sensitive, rapid, and easy-to-use detection of Cs from food and food processing environments is increasing. Here, we developed bioluminescent reporter bacteriophages for viable Cs -specific, substrate-free, rapid detection by introducing luciferase and its corresponding substrate-providing enzyme complex into the virulent phage ΦC01. Although the reporter phage ΦC01_lux, constructed by replacing non-essential genes for phage infectivity with a luxCDABE reporter operon, produced bioluminescence upon Cs infection, the emitted signal was quickly decayed due to the superior bacteriolytic activity of ΦC01. By truncating the membrane pore-forming protein holin and thus limiting its function, the bacterial lysis was delayed and the resultant engineered reporter phage ΦC01_lux_Δhol could produce a more stable and reliable bioluminescent signal. Accordingly, ΦC01_lux_Δhol was able to detect at least an average of 2 CFU/ml of Cs artificially contaminated PIF and Sunsik and food contact surface models within a total of 7 h of assays, including 5 h of pre-enrichment for Cs amplification. The sensitive, easy-to-use, and specific detection of live Cs with the developed reporter phage could be applied as a novel complementary tool for monitoring Cs in food and food-related environments for food safety and public health. [ABSTRACT FROM AUTHOR]

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.

Soft Rot Pectobacteriaceae (SRP; Pectobacterium spp. and Dickeya spp., formerly known as pectinolytic Erwinia spp.) are necrotrophic bacterial pathogens infecting a large number of plant species worldwide, including agriculturally-important crops. Despite the SRP importance in agriculture, little is known about the bacteriophages infecting them, and even less about the prophages present in their genomes. Prophages are recognized as factors underlying bacterial virulence, genomic diversification and ecological fitness that contribute to the novel phenotypic properties of bacterial hosts. Likewise, they are recognized as a driving force of bacterial evolution. In this study, 57 complete genomes of Pectobacterium spp. and Dickeya spp. deposited in NCBI GenBank, were analyzed for the presence of prophage-like elements. Viral sequences were discovered in 95% of bacterial genomes analyzed with the use of PHASTER, PhiSpy, and manual curation of the candidate sequences using NCBI BLAST. In total 37 seemingly intact and 48 putatively defective prophages were found. The 37 seemingly intact prophages (27 sequences in Dickeya spp. genomes and 10 sequences in Pectobacterium spp. genomes) were annotated using RAST. Analysis of the prophage genes encoding viral structural proteins allowed classification of these prophages into different families of the order Caudovirales (tailed bacteriophages) with the SRP prophages of the Myoviridae family (81% of found prophages) being the most abundant. The phylogenetic relationships between prophages were analyzed using amino acid sequences of terminase large subunit (gene terL), integrase (gene int), holin (gene hol), and lysin (gene lys). None of these markers however proved fully useful for clear phylogenetic separation of prophages of SRP into distinct clades. Comparative analyses of prophage proteomes revealed six clusters: five present in Dickeya spp. and one within Pectobacterium spp. When screened for the presence of bacterial genes in the genomes of intact prophages, only one prophage did not contain any ORFs of bacterial origin, the other prophages contained up to 23 genes acquired from bacterial hosts. The bacterial genes present in prophages could possibly affect fitness and virulence of their hosts. The implication of prophage presence in the genomes of Pectobacterium spp. and Dickeya spp. is discussed.

In the cultivation of microalgae for the production of useful compounds, cell disruption to extract the products of interest is a bottleneck process. To establish a cost-effective method to recover these cellular compounds, we developed a method to induce cell lysis via phosphate deficiency in the cyanobacterium Synechocystis sp. PCC 6803. In this system, the promoter from the phoA gene for alkaline phosphatase expressed bacteriophage genes encoding the lytic enzymes holin and endolysin, thus the cell lysis is induced under phosphate-deficient condition. We observed that 90% of the cells, introduced this bacteriophage genes, were lysed after 24 h of incubation under phosphate-deficient conditions. We also developed a method to induce cell lysis in highly concentrated cells for the efficient recovery of valuable cellular products and observed over 90% cell lysis after 16 h of incubation under these conditions. This inducible lysis system may contribute to decreased cell disruption costs in the algal biotechnology industry. [ABSTRACT FROM AUTHOR]

Soft Rot Pectobacteriaceae (SRP; Pectobacterium spp. and Dickeya spp., formerly known as pectinolytic Erwinia spp.) are necrotrophic bacterial pathogens infecting a large number of plant species worldwide, including agriculturally-important crops. Despite the SRP importance in agriculture, little is known about the bacteriophages infecting them, and even less about the prophages present in their genomes. Prophages are recognized as factors underlying bacterial virulence, genomic diversification and ecological fitness that contribute to the novel phenotypic properties of bacterial hosts. Likewise, they are recognized as a driving force of bacterial evolution. In this study, 57 complete genomes of Pectobacterium spp. and Dickeya spp. deposited in NCBI GenBank, were analyzed for the presence of prophage-like elements. Viral sequences were discovered in 95% of bacterial genomes analyzed with the use of PHASTER, PhiSpy, and manual curation of the candidate sequences using NCBI BLAST. In total 37 seemingly intact and 48 putatively defective prophages were found. The 37 seemingly intact prophages (27 sequences in Dickeya spp. genomes and 10 sequences in Pectobacterium spp. genomes) were annotated using RAST. Analysis of the prophage genes encoding viral structural proteins allowed classification of these prophages into different families of the order Caudovirales (tailed bacteriophages) with the SRP prophages of the Myoviridae family (81% of found prophages) being the most abundant. The phylogenetic relationships between prophages were analyzed using amino acid sequences of terminase large subunit (gene terL), integrase (gene int), holin (gene hol), and lysin (gene lys). None of these markers however proved fully useful for clear phylogenetic separation of prophages of SRP into distinct clades. Comparative analyses of prophage proteomes revealed six clusters: five present in Dickeya spp. and one within Pectobacterium spp. When screened for the presence of bacterial genes in the genomes of intact prophages, only one prophage did not contain any ORFs of bacterial origin, the other prophages contained up to 23 genes acquired from bacterial hosts. The bacterial genes present in prophages could possibly affect fitness and virulence of their hosts. The implication of prophage presence in the genomes of Pectobacterium spp. and Dickeya spp. is discussed. [ABSTRACT FROM AUTHOR]

ABSTRACT 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.

When phages infect bacteria cultured in the presence of sublethal doses of antibiotics, the sizes of the phage plaques are significantly increased. This phenomenon is known as phage-antibiotic synergy (PAS). In this study, the observation of PAS was extended to a wide variety of bacterium-phage pairs using different classes of antibiotics. PAS was shown in both Gram-positive and Gram-negative bacteria. Cells stressed with β-lactam antibiotics filamented or swelled extensively, resulting in an increase in phage production. PAS was also sometimes observed in the presence of other classes of antibiotics with or without bacterial filamentation. The addition of antibiotics induced recA expression in various bacteria, but a recA deletion mutant strain of Escherichia coli also showed filamentation and PAS in the presence of quinolone antibiotics. The phage adsorption efficiency did not change in the presence of the antibiotics when the cell surfaces were enlarged as they filamented. Increases in the production of phage DNA and mRNAs encoding phage proteins were observed in these cells, with only a limited increase in protein production. The data suggest that PAS is the product of a prolonged period of particle assembly due to delayed lysis. The increase in the cell surface area far exceeded the increase in phage holin production in the filamented host cells, leading to a relatively limited availability of intracellular holins for aggregating and forming holes in the host membrane. Reactive oxygen species (ROS) stress also led to an increased production of phages, while heat stress resulted in only a limited increase in phage production. IMPORTANCE Phage-antibiotic synergy (PAS) has been reported for a decade, but the underlying mechanism has never been vigorously investigated. This study shows the presence of PAS from a variety of phage-bacterium-antibiotic pairings. We show that increased phage production resulted directly from a lysis delay caused by the relative shortage of holin in filamented bacterial hosts in the presence of sublethal concentrations of stress-inducing substances, such as antibiotics and reactive oxygen species (ROS). [ABSTRACT FROM AUTHOR]

Background: Biocontrol of bacterial pathogens by bacteriophages (phages) represents a promising strategy. Vibrio alginolyticus, a gram-negative bacterium, is a notorious pathogen responsible for the loss of economically important farmed marine animals. To date, few V. alginolyticus phages have been successfully isolated, and only three complete genome sequences of them have been released. The limited available phage resources and poor genomic data hamper research on V. alginolyticus phages and their applications for the biocontrol of V. alginolyticus. Results: We isolated a phage, Vp670, against the V. alginolyticus strain E06333 and obtained its full genomic sequence. It contains 43,121 nucleotides with a GC content of 43.4%, and codes for 49 predicted open reading frames. Observation by electron microscope combined with phylogenetic analysis of DNA polymerase indicates that Vp670 belongs to the subfamily Autographivirinae in the family Podoviridae. orf3 (designated holA) and orf8 (designated cwlQ) are predicted to encode a holin (HolA) and an endolysin (CwlQ), respectively. Expression of holA alone or coexpression of holA and cwlQ from within arrested the growth of Escherichia coli and V. alginolyticus while the expression of cwlQ alone had no effect on the growth of them. Further observation by transmission electron microscopy revealed that the expression of holA vanished the outer membrane and caused the release of cellular contents of V. alginolyticus and the coexpression of holA and cwlQ directly burst the cells and caused a more drastic release of cellular contents. Expression of cwlQ alone in V. alginolyticus did not cause cytomorphological changes. Conclusions: Phage Vp670 is a V. alginolyticus phage belonging to the family of Podoviridae. The genome of Vp670 contains a two-component lysis module, which is comprised of holA and cwlQ. holA is predicted to encode for the holin protein, HolA, and cwlQ is predicted to encode for the endolysin protein, CwlQ. Both holA and cwlQ likely play important roles during the release of phage progeny. [ABSTRACT FROM AUTHOR]

In double-stranded DNA bacteriophages, infection cycles are ended by host cell lysis through the action of phage-encoded endolysins and holins. The precise timing of lysis is regulated by the holin inhibitors, named antiholins. Sequence analysis has revealed that holins with a single transmembrane domain (TMD) are prevalent in Lactobacillus bacteriophages. A temperate bacteriophage of Lactobacillus fermentum, ɸPYB5, has a two-component lysis cassette containing endolysin Lyb5 and holin Hyb5. The hyb5 gene is 465 bp long, encoding 154 amino acid residues with an N-terminal TMD and a large cytoplasmic C-terminal domain. However, the N terminus contains no dual-start motif, suggesting that Hyb5 oligomerization could be inhibited by a specific antiholin. Two internal open reading frames in hyb5, hyb5157-465 and hyb5209-328, were identified as genes encoding putative antiholins for Hyb5 and were coexpressed in trans with lyb5-hyb5 in Escherichia coli. Surprisingly, host cell lysis was delayed by Hyb5157-465 but accelerated by abolishment of the translation initiation site of this protein, indicating that Hyb5157-465 acts as an antiholin to holin Hyb5. Moreover, deletion of 45 amino acid residues at the C terminus of Hyb5 resulted in early cell lysis, even in the presence of Hyb5157-465, implying that the interaction between Hyb5157-465 and Hyb5 occurs at the C terminus of the holin. In vivo and in vitro, Hyb5157-465 and Hyb5 were detected in the cytoplasmic and membrane fractions, respectively, and pulldown assays confirmed direct interaction between Hyb5157-465 and Hyb5. All the results suggest that Hyb5157-465 is an antiholin of Hyb5 that is involved in lysis timing. [ABSTRACT FROM AUTHOR]

The lytic cycle of bacteriophage φ21 for the infected E. coli is initiated by pinholin S21, which determines the timing of host cell lysis through the function of pinholin (S2168) and antipinholin (S2171). The activity of pinholin or antipinholin directly depends on the function of two transmembrane domains (TMDs) within the membrane. For active pinholin, TMD1 externalizes and lies on the surface while TMD2 remains incorporated inside the membrane forming the lining of the small pinhole. In this study, spin labeled pinholin TMDs were incorporated separately into mechanically aligned POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) lipid bilayers and investigated with electron paramagnetic resonance (EPR) spectroscopy to determine the topology of both TMD1 and TMD2 with respect to the lipid bilayer; the TOAC (2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid) spin label was used here because it attaches to the backbone of a peptide and is very rigid. TMD2 was found to be nearly colinear with the bilayer normal (n) with a helical tilt angle of 16 ± 4° while TMD1 lies on or near the surface with a helical tilt angle of 84 ± 4°. The order parameters (~0.6 for both TMDs) obtained from our alignment study were reasonable, which indicates the samples incorporated inside the membrane were well aligned with respect to the magnetic field (B 0). The data obtained from this study supports previous findings on pinholin: TMD1 partially externalizes from the lipid bilayer and interacts with the membrane surface, whereas TMD2 remains buried in the lipid bilayer in the active conformation of pinholin S2168. In this study, the helical tilt angle of TMD1 was measured for the first time. For TMD2 our experimental data corroborates the findings of the previously reported helical tilt angle by the Ulrich group. [Display omitted] • Knowing the topology of bactericidal lytic peptides is important for developing phage therapy. • This study is the first to demonstrate the helical tilt angle of TMD1 of pinholin S2168. • This study corroborates the previously published helical tilt angle for TMD2 of pinholin S2168. [ABSTRACT FROM AUTHOR]

ABSTRACT Huanglongbing (HLB) is a severe disease of citrus caused by an uncultured alphaproteobacterium “Candidatus Liberibacter asiaticus” and transmitted by Asian citrus psyllids (Diaphorina citri). Two prophage genomes, SC1 and SC2, integrated in “Ca. Liberibacter asiaticus” strain UF506 were described previously, and very similar prophages are found resident in the majority of “Ca. Liberibacter asiaticus” strains described worldwide. The SC1 lytic cycle is marked by upregulation of prophage late genes, including a functional holin (SC1_gp110); these late genes are activated when “Ca. Liberibacter asiaticus” is in planta, but not when infecting the psyllid host. We previously reported that the holin promoter is strongly and constitutively active in Liberibacter crescens (a cultured proxy for uncultured “Ca. Liberibacter asiaticus”) but is suppressed in a dose-dependent manner by crude aqueous extracts from D. citri applied exogenously. Here we report that the suppressor activity of the crude psyllid extract was heat labile and abolished by proteinase K treatment, indicating a proteinaceous repressor and of a size smaller than 30 kDa. The repressor was affinity captured from D. citri aqueous extracts using biotinylated holin promoter DNA immobilized on magnetic beads and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein database interrogation was used to identify a small DNA-binding protein encoded by a gene carried by Wolbachia strain wDi, a resident endosymbiont of D. citri as the repressor. The in vitro-translated Wolbachia repressor protein was able to penetrate L. crescens cells, bind to “Ca. Liberibacter asiaticus” promoter DNA, and partially suppress holin promoter-driven β-glucuronidase (GUS) activity, indicating potential involvement of an additional interacting partner(s) or posttranslational modification(s) for complete suppression. Expression of the Wolbachia repressor protein appeared to be constitutive irrespective of “Ca. Liberibacter asiaticus” infection status of the insect host. IMPORTANCE Host acquisition of a new microbial species can readily perturb the dynamics of preexisting microbial associations. Molecular cross talk between microbial associates may be necessary for efficient resource allocation and enhanced survival. Classic examples involve quorum sensing (QS), which detects population densities and is both used and coopted to control expression of bacterial genes, including host adaptation factors. We report that a 56-amino-acid repressor protein made by the resident psyllid endosymbiont Wolbachia can enter cells of Liberibacter crescens, a cultured proxy for the uncultured psyllid endosymbiont “Ca. Liberibacter asiaticus” and repress “Ca. Liberibacter asiaticus” phage lytic cycle genes. Such repression in “Ca. Liberibacter asiaticus” may be critical to survival of both endosymbionts, since phage-mediated lysis would likely breach the immunogenic threshold of the psyllid, invoking a systemic and nonspecific innate immune reaction.

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 coproduction 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. [ABSTRACT FROM AUTHOR]

Monoderm bacteria possess a cell envelope made of a cytoplasmic membrane and a cell wall, whereas diderm bacteria have and extra lipid layer, the outer membrane, covering the cell wall. Both cell types can also produce extracellular protective coats composed of polymeric substances like, for example, polysaccharidic capsules. Many of these structures form a tight physical barrier impenetrable by phage virus particles. Tailed phages evolved strategies/functions to overcome the different layers of the bacterial cell envelope, first to deliver the genetic material to the host cell cytoplasm for virus multiplication, and then to release the virion offspring at the end of the reproductive cycle. There is however a major difference between these two crucial steps of the phage infection cycle: virus entry cannot compromise cell viability, whereas effective virion progeny release requires host cell lysis. Here we present an overview of the viral structures, key protein players and mechanisms underlying phage DNA entry to bacteria, and then escape of the newly-formed virus particles from infected hosts. Understanding the biological context and mode of action of the phage-derived enzymes that compromise the bacterial cell envelope may provide valuable information for their application as antimicrobials.

We have designed a freely accessible program, PhyST, which allows the automated characterization of any family of homologous proteins within the Transporter Classification Database. The program performs an NCBI-PSI-BLAST search and reports (1) the average protein sequence length with standard deviations, (2) the average predicted number of transmembrane segments, (3) the total number of homologues retrieved, (4) a quantitative list of all source phyla, and (5) potential fusion proteins of sizes considerably exceeding the average size of the proteins retrieved. We have applied this program to 58 families of holins, and the results are presented. The results show that holins are very rarely fused to other protein domains, suggesting that holins form transmembrane pores as homooligomers without the participation of other proteins or protein domains. [ABSTRACT FROM AUTHOR]

Genomic architecture is the framework within which genes and regulatory elements evolve and where specific constructs may constrain or potentiate particular adaptations. One such construct is evident in phages that use a headful packaging strategy that results in progeny phage heads packaged with DNA until full rather than encapsidating a simple unit-length genome. Here, we investigate the evolution of the headful packaging phage Sf6 in response to barriers that impede efficient phage adsorption to the host cell. Ten replicate populations evolved faster Sf 6 life cycles by parallel mutations found in a phage lysis gene and/or by large, 1.2-to4.0-kb deletions that remove a mobile genetic IS911 element present in the ancestral phage genome. The fastest life cycles were found in phages that acquired both mutations. No mutations were found in genes encoding phage structural proteins, which were a priori expected from the experimental design that imposed a challenge for phage adsorption by using a Shigella flexneri host lacking receptors preferred by Sf6. We used DNA sequencing, molecular approaches, and physiological experiments on 82 clonal isolates taken from all 10 populations to reveal the genetic basis of the faster Sf 6 life cycle. The majority of our isolates acquired deletions in the phage genome. Our results suggest that deletions are adaptive and can influence the duration of the phage life cycle while acting in conjunction with other lysis time-determining point mutations. [ABSTRACT FROM AUTHOR]

The lysis of bacterial hosts by double-strand DNA bacteriophages, once thought to reflect merely the accumulation of sufficient lysozyme activity during the infection cycle, has been revealed to recently been revealed to be a carefully regulated and temporally scheduled process. For phages of Gramnegative hosts, there are three steps, corresponding to subversion of each of the three layers of the cell envelope: inner membrane, peptidoglycan, and outer membrane. The pathway is controlled at the level of the cytoplasmic membrane. In canonical lysis, a phage encoded protein, the holin, accumulates harmlessly in the cytoplasmic membrane until triggering at an allele-specific time to form micron-scale holes. This allows the soluble endolysin to escape from the cytoplasm to degrade the peptidoglycan. Recently a parallel pathway has been elucidated in which a different type of holin, the pinholin, which, instead of triggering to form large holes, triggers to form small, heptameric channels that serve to depolarize the membrane. Pinholins are associated with SAR endolysins, which accumulate in the periplasm as inactive, membrane-tethered enzymes. Pinholin triggering collapses the proton motive force, allowing the SAR endolysins to refold to an active form and attack the peptidoglycan. Surprisingly, a third step, the disruption of the outer membrane is also required. This is usually achieved by a spanin complex, consisting of a small outer membrane lipoprotein and an integral cytoplasmic membrane protein, designated as o-spanin and i-spanin, respectively. Without spanin function, lysis is blocked and progeny virions are trapped in dead spherical cells, suggesting that the outer membrane has considerable tensile strength. In addition to two-component spanins, there are some single-component spanins, or u-spanins, that have an N-terminal outer-membrane lipoprotein signal and a C-terminal transmembrane domain. A possible mechanism for spanin function to disrupt the outer membrane is to catalyze fusion of the inner and outer membranes. [ABSTRACT FROM AUTHOR]

There have recently been advances in methods for detecting local secondary structures of membrane protein using electron paramagnetic resonance (EPR). A three pulsed electron spin echo envelope modulation (ESEEM) approach was used to determine the local helical secondary structure of the small hole forming membrane protein, S21 pinholin. This ESEEM approach uses a combination of site-directed spin labeling and 2H-labeled side chains. Pinholin S21 is responsible for the permeabilization of the inner cytosolic membrane of double stranded DNA bacteriophage host cells. In this study, we report on the overall global helical structure using circular dichroism (CD) spectroscopy for the active form and the negative-dominant inactive mutant form of S21 pinholin. The local helical secondary structure was confirmed for both transmembrane domains (TMDs) for the active and inactive S21 pinholin using the ESEEM spectroscopic technique. Comparison of the ESEEM normalized frequency domain intensity for each transmembrane domain gives an insight into the α-helical folding nature of these domains as opposed to a π or 3 10 -helix which have been observed in other channel forming proteins. [Display omitted] • A three pulse ESEEM approach is applied to determine the local helical secondary structure of hole forming membrane protein, S21 pinholin. • The local helical secondary structure was confirmed for both transmembrane domains (TMDs) of S21 pinholin using the ESEEM spectroscopic technique. • The use of this ESEEM technique is advantageous over other conventional biophysical structural techniques. [ABSTRACT FROM AUTHOR]