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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]

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]

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. [ABSTRACT FROM AUTHOR]

The genomes of Enterococcus faecalis and Enterococcus faecium bacteriophages were analysed for gene shuffling in the lytic cassettes of bacteriophages infecting. It was found that enterococcal bacteriophages could be classified into well-defined groups based on the size of their genomes and each size group had its own conserved gene composition of lytic cassettes. Enterococcal bacteriophages use a relatively broad spectrum of holins and endolysins with variable cell-wall binding (CWB) and catalytic domains, and most of them utilise a lytic cassette with more than two genes. Enterococcal bacteriophages most commonly use endolysins with amidase catalytic domains and the CWB domain SH3_5. Some bacteriophages possess in their lytic cassette a holin-like gene with the XhlA domain protein, characteristic of hemolysin. Regardless of the shuffling of genes encoding holins and endolysins in lytic modules, a novel example of CWB domain shuffling within enterococcal endolysins was identified. [ABSTRACT FROM AUTHOR]

We have identified a holin-like gene from a goat skin surface metagenome. The ORF designated tmp1 coding for 34 amino acids shared sequence similarity with putative holin-like toxin genes. To analyze the antibacterial activity of tmp1 encoded protein, this ORF was cloned and expressed in Escherichia coli BL21(DE3). The expressed gene product Tmp1 exhibited antibacterial activity against Gram-positive bacteria but not to Gram-negative bacteria. A single transmembrane domain (TMD) was identified within Tmp1 and deletion analysis of the N-terminal region and TMD indicated TMD to be responsible for antibacterial activity. The TMD-dependent antibacterial activity was validated using a synthetic peptide with the amino acid sequence of TMD. Besides antibacterial activity, Tmp1 also complemented the function of holin in a lysis-defective bacteriophage lambda. To broaden the spectrum of antibacterial activity, a mutant library of tmp1 was generated by random mutagenesis. Four mutants with amino acid substitutions at the N-terminus of Tmp1 exhibited increased antibacterial activity against Gram-positive and Gram-negative bacteria and were not hemolytic. An improved activity of these mutant proteins is attributed to their increased hydrophobicity. [ABSTRACT FROM AUTHOR]

The Clostridia produce and secrete Large Clostridial Glucosylating Toxins (LCGTs) responsible for disease symptoms, but the secretion mechanism is largely unknown. Recently, a holin-like protein was shown to be essential for toxin secretion. Holins, typically bacteriophage-specific proteins, are part of the holin-endo(lysin) system that releases phage progeny. To determine if the clostridia also use a lysin, we investigated two conserved putative lysins, M7404_01910 and M7404_02200, in the release of the LCGTs TcdA and TcdB from a Clostridioides difficile ribotype 027 strain, M7404. Sequence analysis and structural modelling indicates that both proteins are related to N-acetylmuramoyl-l-alanine amidases, similar to CD27L, a lysin from the C. difficile phage ΦCD27. Disruption of these genes reveal that only M7404_02200 contributes to toxin secretion and does so in a non-lytic fashion. Peptidoglycan hydrolysis assays show that recombinant M7404_02200 is an active peptidoglycan amidase, confirming its role in TcdA and TcdB secretion in C. difficile M7404. Identification of an N-acetylmuramoyl-l-alanine amidase in the ribotype 027 C. difficile strain M7404, involved in the active secretion of the large clostridial glucosylating toxins, TcdA and TcdB. [ABSTRACT FROM AUTHOR]

An open reading frame encoding a 71-amino acid BhlA bacteriocin-related holin-like peptide was present upstream of 86-amino acid holin-like peptide, xhlB, encoding gene in the genome of Bacillus pumilus strain WAPB4. Analysis of BhlA using TMHMM server suggested one putative transmembrane domain at the N-terminal part and a number of highly charged amino acid residues at the C-terminal part. XhlB of B. pumilus strain WAPB4 composed of two putative transmembrane domains separated by a β-turn, and numerous charged residues in the C-terminus. The dual start motifs were found in both BhlA and XhlB. Structural analysis of their sequence revealed features characteristic for holin. To analyze the effect of BhlA on bacteria cell, its ORF was cloned and expressed in Escherichia coli BL21(DE3). Expression of holin-like peptide, BhlA, was found to be toxic to the host cell. The site of action of BhlA is on the cell membrane and caused bacterial death by cell membrane disruption as clearly demonstrated by transmission electron microscopy or TEM. [ABSTRACT FROM AUTHOR]

Development of novel antibacterial agents is required to control infection with multidrug-resistant Streptococcus suis. HolSMP and LySMP, the holin and lysin of S. suis serotype 2 bacteriophage, named SMP, are responsible for lysis of host cells and release of progeny phage. HolSMP and LySMP expressed in Escherichia coli BL21(DE3) exerted efficient activity at 37 °C, pH 5.2, with addition of 0.8 % β-mercaptoethanol. Lytic spectra of purified HolSMP, LySMP or HolSMP + LySMP mixture were investigated. HolSMP, exhibiting a narrow lytic spectrum, was effective against Staphylococcus aureus and Bacillus subtilis, which were insensitive to LySMP. Moreover, HolSMP was identified as a promising antibacterial agent which was able to extend the spectrum of LySMP. The data suggest that combined use of holin and lysin could be a candidate strategy for resolution of drug resistance. [ABSTRACT FROM AUTHOR]

BhlA, a putative holin-like protein of Bacillus licheniformis AnBa9 expressed in Escherichia coli BL21(DE3) showed antibacterial activity against several gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and Micrococcus luteus. Deletion analysis of bhlA suggests that a hydrophobic transmembrane domain, BhlATM is essential for antibacterial activity. Though the minimum inhibitory concentration (MIC) of BhlA was sevenfold lower than BhlATM, transmembrane domain deleted construct (BhlA∆TM) had no antibacterial activity. The expression of BhlA in E. coli was found to be toxic to cells. Therefore, the bhlA was cloned in yeast surface display vector pYD1 and expressed in Saccharomyces cerevisiae. The surface displayed yeast showed inhibition of several gram-positive bacteria. This recombinant yeast expressing BhlA may be used as biodrug for efficient control of multiple drug-resistant bacterial infections. [ABSTRACT FROM AUTHOR]

An open reading frame encoding an 88 amino acid protein was present downstream of the previously characterized endolysin of Streptomyces aureofaciens phage µ1/6. Structural analysis of its sequence revealed features characteristic for holin. This open reading frame encoding the putative holin was amplified by polymerase chain reaction and cloned into the expression vector pET-21d(+). Synthesis of the holin-like protein resulted in bacterial cell death but not lysis. The holµ1/6 gene was able to complement the defective λ S allele in the nonsuppressing Escherichia coli HB101 strain to produce phage progeny. This fact suggests that the proteins encoded by both phage genes have analogous function, i.e. the streptomycete holin induces nonspecific lesions in the cytoplasmic membrane, through which the λ endolysin gains an access to its substrate, the cell wall. The concomitant expression of both S. aureofaciens holµ1/6 and λ endolysin in E. coli resulted in abrupt cell lysis. This result provided further evidence that the product of holµ1/6 gene is a holin. [ABSTRACT FROM AUTHOR]

Phage λ hybrids were constructed by inserting the t gene of phage T4 in place of the λ holin gene, S. Induction of the hybrid phage resulted in lysis that was just as abrupt as, but occurred much earlier in the vegetative cycle than, that obtained with λ, indicating that t is indeed a holin gene. Moreover, it was possible to impose lysis inhibition (LIN) on induction of the hybrid phage, but not of the parental λ phage, by superinfection with LIN-competent T4. The imposition of the LIN state was found to depend on the allelic state of the rI and t genes of the superinfecting T4 phage, indicating that the LIN-sensitive state of the T holin is transient. Finally, induction of lysogens carrying both holin genes was shown to result in earlier triggering of lysis than with either holin gene alone. This result suggests that the two very dissimilar holins contribute additively to the physiology of the timing mechanism, or, less likely, that they interact to form one mass-action pool. In either case, these results imply a common pathway for holin timing and function. [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]

Bacteriophage-λ-induced host-cell lysis requires two phage-encoded proteins, the S holin and the R transglycosylase. At a specific time during infection, the holin forms a lesion in the cytoplasmic membrane that permits access of the R protein to its substrate, the peptidoglycan. The λ S gene represents the prototype of holin genes with a dual-start motif; they encode two proteins, a lysis effector and a lysis inhibitor. Although the two S proteins differ only by two amino acids (Met-1 and Lys-2) at the N-terminus, the longer product (S107) acts as an inhibitor of the lysis effector (S105). The functional difference between the proteins has been previously ascribed to the Lys-2 residue in S107. It was therefore of interest to determine the subcellular localization of the N-terminus of either S protein. To study the membrane topology of the S proteins, we used the topology probe TEM β-lactamase and an N-terminal tag derived from the Pseudomonas aeruginosa phage Pf3 coat protein. We show that both S proteins have a type III (Nout/Cin) topology. The results provide insight into the regulatory mechanism imposed by the dual-start motif and will be discussed in terms of a model for temporal regulation of the S-dependent “hole” in the membrane. [ABSTRACT FROM AUTHOR]

Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria by bacteriophage ɸX174 gene E mediated lysis. They represent a novel inactivated vaccine platform; however, the practical application of BGs for human vaccines seems to be limited due to the safety concerns on the presence of viable cells in BGs. Therefore, to improve the lysis efficiency of the gene E, we exploited the peptidoglycan hydrolyzing ability of the λ phage holin-endolysins to expedite the process of current BG production system. In this report, we constructed a novel ghost plasmid encoding protein E and holin-endolysins in tandem. We observed that sequential expressions of the gene E and the holin-endolysins elicited rapid and highly efficient Salmonella lysis compared to the lysis mediated by gene E only. These lysed BGs displayed improved immunogenicity in mice compared to the gene E mediated BGs. Consequently, seventy percent of the mice immunized with these novel ghosts survived against a lethal challenge while all the mice vaccinated with gene E mediated ghosts died by day 9 post-infection. We conclude that this novel strategy has the potential to generate highly efficient inactivated candidate vaccines that could replace the currently available bacterial vaccines. [ABSTRACT FROM AUTHOR]

Phages exert profound evolutionary pressure on bacteria by interacting with receptors on the cell surface to initiate infection. While the majority of phages use chromosomally encoded cell surface structures as receptors, plasmid-dependent phages exploit plasmid-encoded conjugation proteins, making their host range dependent on horizontal transfer of the plasmid. Despite their unique biology and biotechnological significance, only a small number of plasmid-dependent phages have been characterized. Here we systematically search for new plasmid-dependent phages targeting IncP and IncF plasmids using a targeted discovery platform, and find that they are common and abundant in wastewater, and largely unexplored in terms of their genetic diversity. Plasmid-dependent phages are enriched in non-canonical types of phages, and all but one of the 65 phages we isolated were non-tailed, and members of the lipid-containing tectiviruses, ssDNA filamentous phages or ssRNA phages. We show that plasmid-dependent tectiviruses exhibit profound differences in their host range which is associated with variation in the phage holin protein. Despite their relatively high abundance in wastewater, plasmid-dependent tectiviruses are missed by metaviromic analyses, underscoring the continued importance of culture-based phage discovery. Finally, we identify a tailed phage dependent on the IncF plasmid, and find related structural genes in phages that use the orthogonal type 4 pilus as a receptor, highlighting the evolutionarily promiscuous use of these distinct contractile structures by multiple groups of phages. Taken together, these results indicate plasmid-dependent phages play an under-appreciated evolutionary role in constraining horizontal gene transfer via conjugative plasmids. Some phages use plasmid-encoded conjugation proteins as receptors to infect their bacterial hosts, making their host range dependent on horizontal transfer of the plasmid. Here, the authors present a method for identification of new plasmid-dependent phages, and find that they are common and abundant in wastewater and their genetic diversity is largely unexplored. [ABSTRACT FROM AUTHOR]

Clostridioides difficile causes a wide range of intestinal diseases through the action of two main cytotoxins, TcdA and TcdB. Ingested spores germinate in the intestine establishing a population of cells that produce toxins and spores. The pathogenicity locus, PaLoc, comprises several genes, including those coding for TcdA/B, for the holin-like TcdE protein, and for TcdR, an auto-regulatory RNA polymerase sigma factor essential for tcdA/B and tcdE expression. Here we show that tcdR, tcdA, tcdB and tcdE are expressed in a fraction of the sporulating cells, in either the whole sporangium or in the forespore. The whole sporangium pattern is due to protracted expression initiated in vegetative cells by σD, which primes the TcdR auto-regulatory loop. In contrast, the forespore-specific regulatory proteins σG and SpoVT control TcdR production and tcdA/tcdB and tcdE expression in this cell. We detected TcdA at the spore surface, and we show that wild type and ΔtcdA or ΔtcdB spores but not ΔtcdR or ΔtcdA/ΔtcdB spores are cytopathic against HT29 and Vero cells, indicating that spores may serve as toxin-delivery vehicles. Since the addition of TcdA and TcdB enhance binding of spores to epithelial cells, this effect may occur independently of toxin production by vegetative cells. This study shows that the genes coding for the two main toxins produced by Clostridioides difficile are expressed during sporulation and that the toxins associate with the mature spores, which have a cytopathic effect on human intestinal cell lines. [ABSTRACT FROM AUTHOR]

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

Lysis cassette genes from phages determine the final lytic event of the host cells. The lysis cassette genes are conserved in phages and prophages. The membrane associated holin from DLP12 prophage, available as a GFP fusion construct, was shown to be overexpressed, using confocal microscopy analysis, in bacterial cells. The protein expression caused cell death in E. coli AG1 strain suggesting the protein was functional. The His-tag HolinGFP protein was purified using cobalt affinity column and was eluted in the presence of different non-ionic detergents DDM ( n-dodecyl-ß- d-maltoside), LDAO (Lauryldimethylamine-oxide), OG ( n-octyl β- d-glucopyranoside) and CE (dodecyl nonaoxyethylene ether). HolinGFP existed predominantly as a dimer in LDAO in Superdex S200 gel filtration chromatography. Circular dichroism and fluorescence spectroscopy of the fluorescent HolinGFP in all four detergents (CE, DDM, LDAO, and OG) confirmed the folded state. Both dithiobis succinimidyl propionate and gluteraldehyde crosslinking revealed the existence of higher order oligomers and dimers. HolinGFP has been functionally and biophysically characterised and is being explored for crystallographic structure determination. [ABSTRACT FROM AUTHOR]

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

Helicobacter pylori infection can cause a variety of gastrointestinal diseases. In severe cases, there is a risk of gastric cancer. Antibiotics are often used for clinical treatment of H. pylori infections. However, because of antibiotic overuse in recent years and the emergence of multidrug-resistant bacteria, there is an urgent need to develop new treatment methods and drugs to achieve complete eradication of H. pylori. Endolysins and holins encoded by bacterial viruses (i.e., phages) represent a promising avenue of investigation. These lyase-based antibacterial drugs act on the bacterial cell wall to destroy the bacteria. Currently, a type of endolysin that has been studied more frequently acts on the amide bond between peptidoglycans, and holin is a transmembrane protein that can punch holes in the cell membrane. However, as a Gram-negative bacterium, H. pylori possesses a layer of impermeable lipopolysaccharides on the cell wall, which prevents endolysin interaction with the cell wall. Therefore, we designed a genetic linkage between an endolysin enzyme and a holin enzyme with a section of polypeptides (e.g., polycations and hydrophobic peptides) that enable penetration of the outer membrane. These complexes were designated "artilysins" and were efficiently expressed in Escherichia coli. In vitro bacteriostasis experiments showed that the purified artilysins had strong bacteriostatic effects on H. pylori. In addition, the surface of H. pylori was perforated and destroyed, as confirmed by electron microscopy, which was proved that artilysins had bacteriolytic effect on H. pylori. [ABSTRACT FROM AUTHOR]

Vibrio phages have emerged as a potential alternative to antibiotic therapy for treating Vibrio infections. In this study, a lytic Vibrio phage, vB_ValA_R15Z against Vibrio alginolyticus ATCC 17749T, was isolated from an aquatic water sample collected in Xiamen, China. The phage had an icosahedral head (diameter 69 ± 2 nm) and a short, non-contractile tail measuring 16 ± 2 nm. The genome of vB_ValA_R15Z was found to be a double-stranded DNA consisting of 43, 552 bp, containing 54 coding sequences (CDSs) associated with phage packaging, structure, DNA metabolism, lysis and additional functions. The BLASTN results indicated that vB_ValA_R15Z shared less than 90.18% similarity with known phages recorded in the NCBI GenBank database, suggesting that vB_ValA_R15Z was a novel Vibrio phage. Furthermore, phylogenetic analysis revealed that vB_ValA_R15Z belongs to the genus Kaohsiungvirus. In addition, a typical lytic mechanism (holin-endolysim) was found in the genome of vB_ValA_R15Z, while no antibiotic resistance- or virulence factor-related gene was detected. Overall, the study provides valuable insights into the isolation and characterization of vB_ValA_R15Z, highlighting its potential as an effective phage therapy option for combating Vibrio alginolyticus infections. [ABSTRACT FROM AUTHOR]

The release of products from microbial cells is an essential process for industrial scale production of bio-based chemicals. However, traditional methods of cell lysis, e.g., mechanical disruption, chemical solvent extraction, and immobilized enzyme degradation, account for a large share of the total production cost. Thus, an efficient cell lysis system is required to lower the cost. This review has focused on our current knowledge of two cell lysis systems, bacteriophage holin-endolysin system, and lipid enzyme hydrolysis system. These systems are controlled by conditionally inducible regulatory apparatus and applied in microbial production of fatty acids and polyhydroxyalkanoates. Moreover, toxin-antitoxin system is also suggested as alternative for its potential applications in cell lysis. Compared with traditional methods of cell disruption, the inducible cell lysis systems are more economically feasible and easier to control and show a promising perspective in industrial production of bio-based chemicals. [ABSTRACT FROM AUTHOR]

This review addresses the deep interior structure of Mercury. Mercury is thought to consist of similar chemical reservoirs (core, mantle, crust) as the other terrestrial planets, but with a relatively much larger core. Constraints on Mercury’s composition and internal structure are reviewed, and possible interior models are described. Large advances in our knowledge of Mercury’s interior are not only expected from imaging of characteristic surface features but particularly from geodetic observations of the gravity field, the rotation, and the tides of Mercury. The low-degree gravity field of Mercury gives information on the differences of the principal moments of inertia, which are a measure of the mass concentration toward the center of the planet. Mercury’s unique rotation presents several clues to the deep interior. From observations of the mean obliquity of Mercury and the low-degree gravity data, the moments of inertia can be obtained, and deviations from the mean rotation speed (librations) offer an exciting possibility to determine the moment of inertia of the mantle. Due to its proximity to the Sun, Mercury has the largest tides of the Solar System planets. Since tides are sensitive to the existence and location of liquid layers, tidal observations are ideally suited to study the physical state and size of the core of Mercury. [ABSTRACT FROM AUTHOR]

Combating bacterial pathogens has become a global concern especially when the antibiotics and chemical agents are failing to control the spread due to its resistance. Bacteriophages act as a safe biocontrol agent by selectively lysing the bacterial pathogens without affecting the natural beneficial microflora. The present study describes the screening of prominent enteric pathogens NDK1, NDK2, NDK3, and NDK4 (Escherichia, Klebsiella, Enterobacter, and Serratia) mostly observed in domestic wastewater; against which KNP1, KNP2, KNP3, and KNP4 phages were isolated. To analyze their potential role in eradicating enteric pathogens and toxicity issue, these bacteriophages were sequenced using next-generation sequencing and characterized based on its genomic content. The isolated bacteriophages were homologous to Escherichia phage (KNP1), Klebsiella phage (KNP2), Enterobacter phage (KNP3), Serratia phage (KNP4), and belonged to Myoviridae family of Caudovirales except for the unclassified KNP4 phage. Draft genome analysis revealed the presence of lytic enzymes such as holing and lysozyme in KNP1 phage, endolysin in KNP2 phage, and endopeptidase with holin in KNP3 phage. The absence of any lysogenic and virulent genes makes this bacteriophage suitable candidate for preparation of phage cocktail to combat the pathogens present in wastewater. However, KNP4 contained a virulent gene rendering it unsuitable to be used as a biocontrol agent. These findings make the phages (KNP1-KNP3) as a promising alternative for the biocontrol of pathogens in wastewater which is the main culprit to spread these dominated pathogens in different natural water bodies. This study also necessitates for genomic screening of bacteriophages for lysogenic and virulence genes prior to its use as a biocontrol agent. [ABSTRACT FROM AUTHOR]

Bacterial ghosts (BGs) are gram-negative bacteria cell membranes without cytoplasmic content obtained by lysis as a result of cell-wall perforation mediated by the φX174 bacteriophage E protein. Introducing into a lytic plasmid lysis gene from other bacteriophages, which target alternative molecular targets, in addition to the E protein gene of bacteriophage φX174, can lead to an increase in the efficiency of lysis and, in some cases, to an increase in the immunogenicity of preparations. Escherichia coli strains carrying plasmids with various combinations of genes encoding protein E of phage φ174 with cassettes of lytic genes of the "choline–endolysin" systems of phages λ or L-413C, providing different degrees of destruction of the cell wall, were obtained for the subsequent selection of the most promising lytic structures. The formation of E. coli ghosts and the release of cell contents was confirmed by transmission electron microscopy. When the cultivation temperature was increased from 28 to 42°C, the lysis of E. coli cultures DH5α/pEYR'-E, DH5α/pEYR'-Y-K, DH5α/pEYR'-E-Y-K, and DH5α/pEYR'-E-Sam7-R-Rz was observed. Lysis was practically not detected during the growth of the DH5α/pEYR'-S-R-Rz culture, as in the control strain DH5α/pEYR'. The results of the analysis of ultrathin sections of BGs preparations by transmission electron microscopy (TEM) were comparable with the data on optical density and the results of inoculation of induced cultures of engineered strains, and also made it possible to assess the fine structure of bacterial cells carrying various combinations of lytic phage genes. BGs of gram-negative bacteria are promising for the creation of highly effective inactivated candidate vaccines that could replace currently available bacterial heat-inactivated and formol vaccines. [ABSTRACT FROM AUTHOR]

We present a new approach to derive efficient and precise algorithms of interest to computer graphics. This is illustrated here by the case of the circle. It also yields algorithms for straight lines and more infrequent curves, such as segments of exponentials. The method is, in essence, the numerical integration of vector fields using only integers. [ABSTRACT FROM AUTHOR]

Bacteriophages are an important source of novel genetic diversity. Sequencing of phage genomes can reveal new proteins with potential uses in phage therapy and help unravel the diversity of biological mechanisms by which phages take over the machinery of the host during infection. To expand the available collection of phage genomes, we have isolated, sequenced, and assembled the genome sequences of three phages that infect three pathogenic Escherichia coli strains: vB_EcoM_DE15, vB_EcoM_DE16, and vB_EcoM_DE17. Morphological characterization and genomic analysis indicated that all three phages were strictly lytic and free from integrases, virulence factors, toxins, and antimicrobial resistance genes. All three phages contained tRNAs, and especially, vB_EcoM_DE17 contained 25 tRNAs. The genomic features of these phages indicate that natural phages are capable of lysing pathogenic E.coli and have great potential in the biocontrol of bacteria. [ABSTRACT FROM AUTHOR]

Bacteriophage-derived endolysin enzymes play a critical role in disintegration of the host bacterial cell wall and hence have gained considerable attention as possible therapeutics for the treatment of drug-resistant infections. Endolysins can target both dividing and non-dividing cells and given the vital role peptidoglycan plays in bacterial survival, bacteria are less likely to modify it even if continuously exposed to lysins. Hence, probability of bacteria developing resistance to lysins appear bleak. Endolysins from mycobacteriophages offer great potential as alternative therapeutics for the drug-resistant TB. However, considering that a large number of mycobacteriophages have been discovered so far, the information on endolysins come from only a few mycobacteriophages. In this study, we report the structural and functional characterization of endolysins (LysinA and LysinB) encoded by mycobacteriophage PDRPxv which belongs to B1 sub cluster. On in silico analysis, we found LysinA to be a modular protein having peptidase domain at the N-terminal (104 aa), a central amidase domain (174 aa) and the peptidoglycan binding domain (62 aa) at the C-terminal. Additionally, 'H-X-H', which is a conserved motif and characteristic of peptidase domains, and the conserved residues His-His-Asp, which are characteristic of amidase domain were also observed. In LysinB enzyme, a single α/β hydrolase domain having a catalytic triad (Ser-Asp-His) and G-X-S-X-G motif, which are characteristic of the serine esterase enzymes were predicted to be present. Both the enzymes were purified as recombinant proteins and their antimycobacterial activity against M. smegmatis was demonstrated through turbidimetric experiments and biochemical assay. Interesting observation in this study is the secretory nature of LysinA evident by its periplasmic expression in E.coli, which might explain the ability of PDRPxv to lyse the bacterial host in the absence of transmembrane Holin protein. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance is a serious threat to public health around the globe. According to the World Health Organization, there will be a return to the pre-penicillin era by 2050 if no new antimicrobials are discovered. It is therefore necessary to find new antimicrobials and alternatives. Pseudomonas aeruginosa exhibits resistance against many antibiotics and causes a variety of infections in immunocompromised individuals and especially in those with burn wounds and lung infections. Bacteriophage RLP against P. aeruginosa strain PA-1 was isolated from the Ravi River near Lahore. It showed marked stability at different pH values and temperatures, with the maximum storage stability at 4 °C. It demonstrated the ability to inhibit bacterial growth for up to 20 h, replicated in 25 min, and produced 154 virions per infected cell. RLP showed a broad host range, infecting 50% (19/38) of the multiple-drug-resistant (MDR) P. aeruginosa strains that were tested. The 43-kbp-long genome of RLP is a double-stranded DNA molecule that encodes 56 proteins in total: 34 with known functions, and 22 with no homolog in the gene databases. A cascade system of lytic machinery is also present in the form of four genes (R/z, R/z1, holin and endolysin). Therapeutic studies of RLP in bacteremic mice infected with P. aeruginosa strain PA-1 demonstrated a 92% survival rate in the treated group compared with 7.4% in the untreated group, and this result was statistically significant. Based on its physiological and genetic properties, ability to cause a reduction in bacterial growth in vitro and its in vivo therapeutic efficacy, RLP could be a good candidate for use in phage therapy. [ABSTRACT FROM AUTHOR]

Phages are considered the most abundant and diverse biological entities on Earth. Although many phages have been isolated, most of this diversity remains completely unexplored. Recent advances in genome sequencing, however, have provided unprecedented glimpses into the virosphere. The lytic cold-active Siphoviridae bacteriophage VW6S, infecting the host Pseudomonas fluorescens W-6, was isolated from the Napahai plateau wetland in China. The newly sequenced phage VW6S contained double-stranded DNA with a genome size of 37,917 bp, an overall GC content of 56.90% and 52 open reading frames (ORFs). The genome was organized into several modules containing genes for packaging, structural proteins, replication/transcription, and cell lysis. The sequence contained 16 transcription terminators, no tRNAs. The lysis cassette of VW6S consisted of 4 proteins (holin protein, lysin protein and 2 putative spanins). This is the first report on cold-active SiphoviridaePseudomonas fluorescens bacteriophage genome sequencing. [ABSTRACT FROM AUTHOR]

The rapid development of quantum magnonics can be attributed to its potential applications in quantum computing, information processing and the creation of hybrid quantum systems. Quantum quasiparticles, known as magnons, undergo Bose condensation at high densities, a phenomenon observed during radiofrequency excitation. This phenomenon was initially discovered in the antiferromagnetic superfluid 3 He. Magnonic superfluidity effects, Josephson effect and magnon tunnelling were also observed in this system. Similar effects have been identified in yttrium iron garnet at room temperature, suggesting the possibility of utilising these effects in a manner akin to superconductivity. In this article, we conduct simulations of magnon tunnelling processes across the energy gap, aiming to compare these simulations with experimental findings. [ABSTRACT FROM AUTHOR]

The widespread bacterial genus Sulfurimonas is metabolically versatile and occupies a key ecological niche in different habitats, but its interaction with bacteriophages remains unexplored. Here we systematically investigated the genetic diversity, taxonomy and interaction patterns of Sulfurimonas-associated phages based on sequenced microbial genomes and metagenomes. High-confidence phage contigs related to Sulfurimonas were retrieved from various ecosystems, clustered into 61 viral operational taxonomic units across three viral realms, including Duplodnaviria, Monodnaviria and Varidnaviria. Head-tail phages of Caudoviricetes were assigned to 19 genus-level viral clusters, the majority of which were distantly related to known viruses. Notably, diverse double jelly-roll viruses and inoviruses were also linked to Sulfurimonas, representing two commonly overlooked phage groups. Historical and current phage infections were revealed, implying viral impact on the evolution of host adaptive immunity. Additionally, phages carrying auxiliary metabolic genes might benefit hosts by compensating or augmenting sulfur metabolism. This study highlights the diversity and novelty of Sulfurimonas-associated phages with divergent tailless lineages, providing basis for further investigation of phage-host interactions within this genus. A catalog of phages associated with Sulfurimonas reveals unexplored diversity and potential viral impacts, providing new insight into the phage-host coevolution. [ABSTRACT FROM AUTHOR]

Cell motility increases the fitness of bacterial cells. Previous research focused on the transcriptional regulator CdsR, which represses cellular autolysis and promotes spore formation in Bacillus thuringiensis. However, the targets of CdsR are mostly unknown. Here, we reported a new function of CdsR in regulating cell motility. Mutation of cdsR results in increase of cell mobility, and a number of related genes were upregulated compared to wild type HD73. Thus, we investigated the transcription of the fla/che gene cluster, which involves in cell mobility and comprises eight operons/genes, including motAB1, cheY-yrhK, lamB-cheR, yaaR-fliG2, cheV-mogR, hag1, hag2, and yjbJ-flgG. Additionally, the motAB2 operon was discovered, which consists of homologs genes motA2 and motB2 that are like motA1 and motB1. Through promoter-lacZ fusion assays and EMSA experiments, it was discovered that CdsR directly regulates the motAB1, cheY-yrhK, lamB-cheR, yaaR-fliG2, cheV-mogR, hag1, hag2, yjbJ-flgG, and motAB2 operons by binding to their promoter regions. Importantly, it was confirmed that CdsR is a metalloregulator and the binding to promoter can be inhibited by Cu (II) ions. This research enhances our understanding of the regulation of cell mobility in B. thuringiensis. [ABSTRACT FROM AUTHOR]

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]

The major virulence factors of Clostridium difficile are toxins A and B. These toxins are encoded by tcdA and tcdB genes, which form a pathogenicity locus (PaLoc) together with three additional genes that have been implicated in regulation (tcdR and tcdC) and secretion (tcdE). To date, the PaLoc has always been found in the same location and is replaced in non-toxigenic strains by a highly conserved 75/115 bp non-coding region. Here, we show new types of C. difficile pathogenicity loci through the genome analysis of three atypical clinical strains and describe for the first time a variant strain producing only toxin A (A+B-). Importantly, we found that the PaLoc integration sites of these three strains are located in the genome far from the usual single known PaLoc integration site. These findings allowed us to propose a new model of PaLoc evolution in which two "Mono-Toxin PaLoc" sites are merged to generate a single "Bi-Toxin PaLoc". [ABSTRACT FROM AUTHOR]

Photobacterium damselae subsp. damselae (PDD) is a pathogenic bacterium that affects both marine animals and humans. It poses a significant problem for aquaculture and a threat to public health. Phages are considered potential therapeutics due to their high specificity against pathogens and environmental friendliness. The Phage-Antibiotic Synergy (PAS) approach has recently shown strong potential as an alternative strategy to overcome the resistance of bacteria to both phages and antibiotics. In this study, we investigated the genomic characteristics of the TEMp-D1 phage, which specifically infects PDD, and the effects of different environmental conditions on phage stability. The genome of TEMp-D1 is 72,582 bp in length with a GC content of 55.1%. Sequence analysis revealed 97 potential coding sequences (CDS) with an N4-like genomic organization. Phylogenetic analysis using the whole genome proteomic tree and the amino acid sequence of the large terminase subunit showed a close relationship between TEMp-D1 and phages infecting Vibrio and Pseudomonas species. Furthermore, the effects of PAS were examined using sublethal doses (1/4 MIC) of four antibiotics (oxytetracycline, florfenicol, sulfadiazine-trimethoprim, and enrofloxacin) and the TEMp-D1 phage on in vitro PDD biofilm formation and cell growth. Inhibition rates were significantly higher in the PAS-treated groups compared to those treated with phage or antibiotics alone. These results demonstrate the potential of TEMp-D1 and antibiotic combinations for use against PDD in aquaculture and other in vivo applications. [ABSTRACT FROM AUTHOR]

More and more virulent phages that are fundamental materials for phage therapy have been isolated, characterized and categorized on GenBank. Phage ST31 infecting Escherichia coli H21 was isolated from wastewater and sequenced using an Illumina Hiseq system. Opening reading frames were identified using PHASTER and predicted using BLASTp analysis. Genomic analyses revealed that this was a virulent phage containing a circular double-stranded DNA and that the complete genome consisted of 39,693 nucleotides with an average GC content of 49.98 %. This study may provide possible alternative materials for phage therapy. [ABSTRACT FROM AUTHOR]

Typhoidal Salmonella enterica serovars, such as Typhi and Paratyphi A, cause severe systemic infections, thereby posing a significant threat as human-adapted pathogens. This study focuses on cytolysin A (ClyA), a virulence factor essential for bacterial dissemination within the human body. We show that ClyA is exclusively expressed by intracellular S. Paratyphi A within the Salmonella-containing vacuole (SCV), regulated by the PhoP/Q system and SlyA. ClyA localizes in the bacterial periplasm, suggesting potential secretion. Deletion of TtsA, an essential Type 10 Secretion System component, completely abolishes intracellular ClyA detection and its presence in host cell supernatants. Host cells infected with wild-type S. Paratyphi A contain substantial ClyA, with supernatants capable of lysing neighboring cells. Notably, ClyA selectively lyses macrophages and erythrocytes while sparing epithelial cells. These findings identify ClyA as an intracellularly induced cytolysin, dependent on the SCV environment and secreted via a Type 10 Secretion System, with specific cytolytic activity. Typhoidal Salmonella enterica strains cause serious infections in humans. Here, Krone et al show that the bacterial protein cytolysin A is secreted intracellularly where it selectively kills specific cell types and helps to establish bacterial infection. [ABSTRACT FROM AUTHOR]

Infections caused by Gram-negative bacteria are leading causes of mortality worldwide. Due to the rise in antibiotic resistant strains, there is a desperate need for alternative strategies to control infections caused by these organisms. One such approach is the prevention of infection through vaccination. While live attenuated and heat-killed bacterial vaccines are effective, they can lead to adverse reactions. Newer vaccine technologies focus on utilizing polysaccharide or protein subunits for safer and more targeted vaccination approaches. One promising avenue in this regard is the use of proteins released by the Type 5 secretion system (T5SS). This system is the most prevalent secretion system in Gram-negative bacteria. These proteins are compelling vaccine candidates due to their demonstrated protective role in current licensed vaccines. Notably, Pertactin, FHA, and NadA are integral components of licensed vaccines designed to prevent infections caused by Bordetella pertussis or Neisseria meningitidis. In this review, we delve into the significance of incorporating T5SS proteins into licensed vaccines, their contributions to virulence, conserved structural motifs, and the protective immune responses elicited by these proteins. [ABSTRACT FROM AUTHOR]

The escalating antibiotic resistance in mycobacterial species poses a significant threat globally, necessitating an urgent need to find alternative solutions. Bacteriophage-derived endolysins, which facilitate phage progeny release by attacking bacterial cell walls, present promising antibacterial candidates due to their rapid lytic action, high specificity and low risk of resistance development. In mycobacteria, owing to the complex, hydrophobic cell wall, mycobacteriophages usually synthesize two endolysins: LysinA, which hydrolyzes peptidoglycan; LysinB, which delinks mycolic acid-containing outer membrane and arabinogalactan, releasing free mycolic acid. In this study, we conducted domain analysis and functional characterization of a novel LysinB from RitSun, an F2 sub-cluster mycobacteriophage from our phage collection. Several key properties of RitSun LysinB make it an important antimycobacterial agent: its ability to lyse Mycobacterium smegmatis from without, a higher than previously reported specific activity of 1.36 U/mg and its inhibitory effect on biofilm formation. Given the impermeable nature of the mycobacterial cell envelope, dissecting RitSun LysinB at the molecular level to identify its cell wall-destabilizing sequence could be utilized to engineer other native lysins as fusion proteins, broadening their activity spectrum. [ABSTRACT FROM AUTHOR]

Stutzerimonas have been extensively studied due to their remarkable metabolic and physiological diversity. However, research on its phages is currently limited. In this study, we isolated a novel double-stranded DNA (dsDNA) phage, vB_SstM-PG1, from the marine environment that infects Stutzerimonas stutzeri G1. Its dsDNA genome is 37204 bp long with a G/C content of 64.14% and encodes 54 open reading frames. The phage possesses a tail packaging structure that is different from known Stutzerimonas stutzeri phages and exhibits structural protein characteristics similar to those of temperate phages. In addition, two genes of toxin-antitoxin system, including YdaS_antitoxin and HEPN_SAV_6107, were found in the vB_SstM-PG1 genome and play important roles in regulating host growth and metabolism. With phylogenetic tree and comparative genomic analysis, it has been determined that vB_ SstM-PG1 is not closely related to any phages previously identified in the GenBank database. Instead, it has a connection with enigmatic, uncultured viruses. Specifically, the vB_SstM-PG1 virus exhibits an average nucleotide identity of over 70% with six uncultivated viruses identified in the IMG/VR v4 database. This significant finding has resulted in the identification of a novel viral genus known as Metabovirus. [ABSTRACT FROM AUTHOR]

Psyllid species, including the potato psyllid (PoP) Bactericera cockerelli (Sulc) (Triozidae) serve as host and vector of “Candidatus Liberibacter spp.” (“Ca. Liberibacter”), which also infects diverse plant hosts, including citrus and tomato. Psyllid transmission of “Ca. Liberibacter” is circulative and propagative. The time of “Ca. Liberibacter” acquisition and therefore vector life stage most competent for bacterial transmission varies by pathosystems. Here, the potato psyllid-“Ca. Liberibacter solanacearum” (CLso) pathosystem was investigated to dissect CLso-prophage interactions in the tomato plant and PoP-psyllid host by real-time quantitative reverse transcriptase amplification of CLso genes/loci with predicted involvement in host infection and psyllid-CLso transmission. Genes/loci analyzed were associated with (1) CLso-adhesion, -invasion, -pathogenicity, and -motility, (2) prophage-adhesion and pathogenicity, and (3) CLso-lysogenic cycle. Relative gene expression was quantified by qRT-PCR amplification from total RNA isolated from CLso-infected 1st–2nd and 4th–5th nymphs and teneral adults and CLso-infected tomato plants in which CLso infection is thought to occur without SC1-SC2 replication. Gene/loci expression was host-dependent and varied with the psyllid developmental stage. Loci previously associated with repressor-anti-repressor regulation in the “Ca Liberibacter asiaticus”-prophage pathosystem, which maintains the lysogenic cycle in Asian citrus psyllid Diaphorina citri, were expressed in CLso-infected psyllids but not in CLso-infected tomato plants. [ABSTRACT FROM AUTHOR]

The escalating incidence of foodborne salmonellosis poses a significant global threat to food safety and public health. As antibiotic resistance in Salmonella continues to rise, there is growing interest in bacteriophages as potential alternatives. In this study, we isolated, characterized, and evaluated the biocontrol efficacy of lytic phage L223 in chicken meat. Phage L223 demonstrated robust stability across a broad range of temperatures (20–70 °C) and pH levels (2–11) and exhibited a restricted host range targeting Salmonella spp., notably Salmonella Typhimurium and Salmonella Enteritidis. Characterization of L223 revealed a short latent period of 30 min and a substantial burst size of 515 PFU/cell. Genomic analysis classified L223 within the Caudoviricetes class, Guernseyvirinae subfamily and Jerseyvirus genus, with a dsDNA genome size of 44,321 bp and 47.9% GC content, featuring 72 coding sequences devoid of antimicrobial resistance, virulence factors, toxins, and tRNA genes. Application of L223 significantly (p < 0.005) reduced Salmonella Typhimurium ATCC 14,028 counts by 1.24, 2.17, and 1.55 log CFU/piece after 2, 4, and 6 h of incubation, respectively, in experimentally contaminated chicken breast samples. These findings highlight the potential of Salmonella phage L223 as a promising biocontrol agent for mitigating Salmonella contamination in food products, emphasizing its relevance for enhancing food safety protocols. [ABSTRACT FROM AUTHOR]