Construction of Leaderless-Bacteriocin-Producing Bacteriophage Targeting E. coli and Neighboring Gram-Positive Pathogens.

Lytic bacteriophages are expected as effective tools to control infectious bacteria in human and pathogenic or spoilage bacteria in foods. Leaderless bacteriocins (LLBs) are simple bacteriocins produced by Gram-positive bacteria. LLBs do not possess an N-terminal leader peptide in the precursor, which means that they are active immediately after translation. In this study, we constructed a novel antimicrobial agent, an LLB-producing phage (LLB-phage), by genetic engineering to introduce the LLB structural gene into the lytic phage genome. To this end, lnqQ (structure gene of an LLB, lacticin Q) and trxA, an essential gene for T7 phage genome replication, were integrated in tandem into T7 phage genome using homologous recombination in Escherichia coli host strain. The recombinant lnqQ-T7 phage was isolated by a screening method using ΔtrxA host strain. lnqQ-T7 phage formed a clear halo in agar plates containing both E. coli and lacticin Q-susceptible Bacillus coagulans, indicating that lnqQ-T7 phage could produce a significant amount of lacticin Q. Lacticin Q production did not exert a significant effect on the lytic cycle of T7 phage. In fact, the production of lacticin Q enhanced T7 phage lytic activity and helped to prevent the emergence of bacterial populations resistant against this phage. These results serve as a proof of principle for LLB-phages. There are different types of LLBs and phages, meaning that in the future, it may be possible to produce any number of LLB-phages which can be designed to efficiently control different types of bacterial contamination in different settings. IMPORTANCE We demonstrated that we could combine LLB and phage to construct promising novel antimicrobial agents, LLB-phage. The first LLB-phage, lnqQ-T7 phage, can control the growth of both the Gram-negative host strain and neighboring Gram-positive bacteria while preventing the emergence of phage resistance in the host strain. There are several different types of LLBs and phages, suggesting that we may be able to design a battery of LLB-phages by selecting novel combinations of LLBs and phages. These constructs could be tailored to control various bacterial contaminations and infectious diseases.

Crystallographic Structure Determination of Bacteriophage Endolysins.

Bacteriophages produce endolysins that target and cleave the hosts peptidoglycan to release their progeny at the end of the infection cycle. These proteins can be used for the eradication of pathogenic bacteria, but also for their detection. Endolysins may contain a single catalytic domain or several domains, including a cell wall binding domain. To understand their function in detail and design mutated or chimeric molecules with novel properties, knowledge of their structures and detailed mechanisms is necessary. X-ray protein crystallography is an excellent method to obtain high-resolution structures of biological macromolecules, and here we describe the method and the folds of known endolysin domains.

Structures of the tailed bacteriophages that infect Gram-positive bacteria.

Productive virus infection depends upon delivery of viral genomic material into the host cell cytoplasm. The tails of bacteriophages recognize host cells and mediate host cell wall and membrane penetration. Recent cryo-electron microscopy studies have revealed near atomic-resolutions structures of the entire or almost entire bacteriophage particles of model systems including phi29, P22, P68, and T4. These structures allow comparisons between not only different states of the same phage but also between distantly related phages. In this review, we summarize the findings from recent structural studies of the bacteriophages that target Gram-positive bacteria, for a better understanding of the interactions between host cells and bacteriophages.

The mannose phosphotransferase system (Man-PTS) - Mannose transporter and receptor for bacteriocins and bacteriophages.

Mannose transporters constitute a superfamily (Man-PTS) of the Phosphoenolpyruvate Carbohydrate Phosphotransferase System (PTS). The membrane complexes are homotrimers of protomers consisting of two subunits, IIC and IID. The two subunits without recognizable sequence similarity assume the same fold, and in the protomer are structurally related by a two fold pseudosymmetry axis parallel to membrane-plane (Liu et al. (2019) Cell Research 29 680). Two reentrant loops and two transmembrane helices of each subunit together form the N-terminal transport domain. Two three-helix bundles, one of each subunit, form the scaffold domain. The protomer is stabilized by a helix swap between these bundles. The two C-terminal helices of IIC mediate the interprotomer contacts. PTS occur in bacteria and archaea but not in eukaryotes. Man-PTS are abundant in Gram-positive bacteria living on carbohydrate rich mucosal surfaces. A subgroup of IICIID complexes serve as receptors for class IIa bacteriocins and as channel for the penetration of bacteriophage lambda DNA across the inner membrane. Some Man-PTS are associated with host-pathogen and -symbiont processes.

Beyond the CRISPR-Cas safeguard: PICI-encoded innate immune systems protect bacteria from bacteriophage predation.

Phage satellites are genetic elements that depend on helper phages for induction, packaging and transfer. To promote their lifestyles, they have evolved elegant and sophisticated strategies to inhibit phage reproduction, which will be reviewed here. We will principally focus on the convergent interference mechanisms used by phage-inducible chromosomal islands (PICIs), which are a family of satellite phages present in both Gram-positive and Gram-negative bacteria. While some PICI elements have been extensively studied for their roles in virulence and antibiotic resistance, recent studies have highlighted their relevance in controlling phage ecology and diversity. In many cases, these interference mechanisms are complemented by additional strategies that promote the preferential PICI packaging and dissemination of these elements in nature. Since the PICI-encoded mechanisms target conserved phage mechanisms, we propose here that the PICIs form part of the initial innate immune system that phages must overcome to infect their bacterial host.

Dip-a-Dee-Doo-Dah: Bacteriophage-Mediated Rescoring of a Harmoniously Orchestrated RNA Metabolism.

RNA turnover and processing in bacteria are governed by the structurally divergent but functionally convergent RNA degradosome, and the mechanisms have been researched extensively in Gram-positive and Gram-negative bacteria. An emerging research field focuses on how bacterial viruses hijack all aspects of the bacterial metabolism, including the host machinery of RNA metabolism. This review addresses research on phage-based influence on RNA turnover, which can act either indirectly or via dedicated effector molecules that target degradosome assemblies. The structural divergence of host RNA turnover mechanisms likely explains the limited number of phage proteins directly targeting these specialized, host-specific complexes. The unique and nonconserved structure of DIP, a phage-encoded inhibitor of the Pseudomonas degradosome, illustrates this hypothesis. However, the natural occurrence of phage-encoded mechanisms regulating RNA turnover indicates a clear evolutionary benefit for this mode of host manipulation. Further exploration of the viral dark matter of unknown phage proteins may reveal more structurally novel interference strategies that, in turn, could be exploited for biotechnological applications.

Phage-Antibiotic Synergy via Delayed Lysis.

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 ß-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).

Molecular Basis of Bacterial Host Interactions by Gram-Positive Targeting Bacteriophages.

The inherent ability of bacteriophages (phages) to infect specific bacterial hosts makes them ideal candidates to develop into antimicrobial agents for pathogen-specific remediation in food processing, biotechnology, and medicine (e.g., phage therapy). Conversely, phage contaminations of fermentation processes are a major concern to dairy and bioprocessing industries. The first stage of any successful phage infection is adsorption to a bacterial host cell, mediated by receptor-binding proteins (RBPs). As the first point of contact, the binding specificity of phage RBPs is the primary determinant of bacterial host range, and thus defines the remediative potential of a phage for a given bacterium. Co-evolution of RBPs and their bacterial receptors has forced endless adaptation cycles of phage-host interactions, which in turn has created a diverse array of phage adsorption mechanisms utilizing an assortment of RBPs. Over the last decade, these intricate mechanisms have been studied intensely using electron microscopy and X-ray crystallography, providing atomic-level details of this fundamental stage in the phage infection cycle. This review summarizes current knowledge surrounding the molecular basis of host interaction for various socioeconomically important Gram-positive targeting phage RBPs to their protein- and saccharide-based receptors. Special attention is paid to the abundant and best-characterized Siphoviridae family of tailed phages. Unravelling these complex phage-host dynamics is essential to harness the full potential of phage-based technologies, or for generating novel strategies to combat industrial phage contaminations.

Extending the hosts of Tectiviridae into four additional genera of Gram-positive bacteria and more diverse Bacillus species.

Tectiviridae are composed of tailless bacteriophages with an icosahedral capsid and an inner membrane enclosing a double-stranded 15 kb linear DNA genome. Five of the seven previously studied Tectivirus isolates infect bacteria from Bacillus cereus sensu lato group (Betatectivirus), one distantly related member (PRD1) infect Enterobactericeae (Alpatectivirus) and one recently discovered virus infect Gluconobacter cerinus (Gammatectivirus). Here we expand the host spectrum of Betatectivirus elements to four additional genera (Streptococcus, Exiguobacterium, Clostridium and Brevibacillus) and to more distantly related Bacillus species (B. pumilus and B. flexus) by studying the genomes of fourteen novel tectiviral elements. Overall, the genomes show significant conservation in gene synteny and in modules responsible for genome replication and formation of the virion core (including DNA packaging). Notable variation exists in regions encoding host attachment and lysis along with the surrounding area of a site in which mutations are known to alter phage life cycle.

Cross-genus rebooting of custom-made, synthetic bacteriophage genomes in L-form bacteria.

Engineered bacteriophages provide powerful tools for biotechnology, diagnostics, pathogen control, and therapy. However, current techniques for phage editing are experimentally challenging and limited to few phages and host organisms. Viruses that target Gram-positive bacteria are particularly difficult to modify. Here, we present a platform technology that enables rapid, accurate, and selection-free construction of synthetic, tailor-made phages that infect Gram-positive bacteria. To this end, custom-designed, synthetic phage genomes were assembled in vitro from smaller DNA fragments. We show that replicating, cell wall-deficient Listeria monocytogenes L-form bacteria can reboot synthetic phage genomes upon transfection, i.e., produce virus particles from naked, synthetic DNA. Surprisingly, Listeria L-form cells not only support rebooting of native and synthetic Listeria phage genomes but also enable cross-genus reactivation of Bacillus and Staphylococcus phages from their DNA, thereby broadening the approach to phages that infect other important Gram-positive pathogens. We then used this platform to generate virulent phages by targeted modification of temperate phage genomes and demonstrated their superior killing efficacy. These synthetic, virulent phages were further armed by incorporation of enzybiotics into their genomes as a genetic payload, which allowed targeting of phage-resistant bystander cells. In conclusion, this straightforward and robust synthetic biology approach redefines the possibilities for the development of improved and completely new phage applications, including phage therapy.

Bacteriophages and bacteriophage-derived endolysins as potential therapeutics to combat Gram-positive spore forming bacteria.

Since their discovery in 1915, bacteriophages have been routinely used within Eastern Europe to treat a variety of bacterial infections. Although initially ignored by the West due to the success of antibiotics, increasing levels and diversity of antibiotic resistance is driving a renaissance for bacteriophage-derived therapy, which is in part due to the highly specific nature of bacteriophages as well as their relative abundance. This review focuses on the bacteriophages and derived lysins of relevant Gram-positive spore formers within the Bacillus cereus group and Clostridium genus that could have applications within the medical, food and environmental sectors.

The Effect of Bacteriophage Preparations on Intracellular Killing of Bacteria by Phagocytes.

Intracellular killing of bacteria is one of the fundamental mechanisms against invading pathogens. Impaired intracellular killing of bacteria by phagocytes may be the reason of chronic infections and may be caused by antibiotics or substances that can be produced by some bacteria. Therefore, it was of great practical importance to examine whether phage preparations may influence the process of phagocyte intracellular killing of bacteria. It may be important especially in the case of patients qualified for experimental phage therapy (approximately half of the patients with chronic bacterial infections have their immunity impaired). Our analysis included 51 patients with chronic Gram-negative and Gram-positive bacterial infections treated with phage preparations at the Phage Therapy Unit in Wroclaw. The aim of the study was to investigate the effect of experimental phage therapy on intracellular killing of bacteria by patients' peripheral blood monocytes and polymorphonuclear neutrophils. We observed that phage therapy does not reduce patients' phagocytes' ability to kill bacteria, and it does not affect the activity of phagocytes in patients with initially reduced ability to kill bacteria intracellularly. Our results suggest that experimental phage therapy has no significant adverse effects on the bactericidal properties of phagocytes, which confirms the safety of the therapy.

Transposable Mu-like phages in Firmicutes: new instances of divergence generating retroelements.

Sequences of the most conserved proteins encoded by transposable (pro)phages were used to search recently sequenced Firmicute genomes for candidate transposable prophages. One complete Mu-like prophage, SglyMu-1, was identified, in four copies, in the Syntrophobotulus glycolicus DSM 8271 chromosome sequence. Related prophages were also found in partially assembled genomic sequences of other Firmicutes and newly sequenced Proteobacteria genomes, opening the road to the use of Mu-like derived genetic tools in Gram(+) bacteria. SglyMu-1 appears to carry a host variation system related to the DGR tropism switching retroelements, first characterized in Bordetella phages BPP-1, BIP-1 and BMP-1. Transposable phages are thus thriving among Firmicutes and can harness either of two host variation systems, the fiber genes inversion and reverse transcriptase-mediated site-directed, adenine-specific mutagenesis.

Bacteriophage electron microscopy.

Since the advent of the electron microscope approximately 70 years ago, bacterial viruses and electron microscopy are inextricably linked. Electron microscopy proved that bacteriophages are particulate and viral in nature, are complex in size and shape, and have intracellular development cycles and assembly pathways. The principal contribution of electron microscopy to bacteriophage research is the technique of negative staining. Over 5500 bacterial viruses have so far been characterized by electron microscopy, making bacteriophages, at least on paper, the largest viral group in existence. Other notable contributions are cryoelectron microcopy and three-dimensional image reconstruction, particle counting, and immunoelectron microscopy. Scanning electron microscopy has had relatively little impact. Transmission electron microscopy has provided the basis for the recognition and establishment of bacteriophage families and is one of the essential criteria to classify novel viruses into families. It allows for instant diagnosis and is thus the fastest diagnostic technique in virology. The most recent major contribution of electron microscopy is the demonstration that the capsid of tailed phages is monophyletic in origin and that structural links exist between some bacteriophages and viruses of vertebrates and archaea. DNA sequencing cannot replace electron microscopy and vice versa.

Role of CRISPR/cas system in the development of bacteriophage resistance.

Acquisition of foreign DNA can be of advantage or disadvantage to the host cell. New DNAs can increase the fitness of an organism to certain environmental conditions; however, replication and maintenance of incorporated nucleotide sequences can be a burden for the host cell. These circumstances have resulted in the development of certain cellular mechanisms limiting horizontal gene transfer, including the immune system of vertebrates or RNA interference mechanisms in eukaryotes. Also, in prokaryotes, specific systems have been characterized, which are aimed especially at limiting the invasion of bacteriophage DNA, for example, adsorption inhibition, injection blocking, restriction/modification, or abortive infection. Quite recently, another distinct mechanism limiting horizontal transfer of genetic elements has been identified in procaryotes and shown to protect microbial cells against exogenous nucleic acids of phage or plasmid origin. This system has been termed CRISPR/cas and consists of two main components: (i) the CRISPR (clustered, regularly interspaced short palindromic regions) locus and (ii) cas genes, encoding CRISPR-associated (Cas) proteins. In simplest words, the mechanism of CRISPR/cas activity is based on the active integration of small fragments (proto-spacers) of the invading DNAs (phage or plasmids) into microbial genomes, which are subsequently transcribed into short RNAs that direct the degradation of foreign invading DNA elements. In this way, the host organism acquires immunity toward mobile elements carrying matching sequences. The CRISPR/cas system is regarded as one of the earliest defense system that has evolved in prokaryotic organisms. It is inheritable, but at the same time is unstable when regarding the evolutionary scale. Comparative sequence analyses indicate that CRISPR/cas systems play an important role in the evolution of microbial genomes and their predators, bacteriophages.

Pseudolysogeny.

Pseudolysogeny can be defined as the stage of stalled development of a bacteriophage in a host cell without either multiplication of the phage genome (as in lytic development) or its replication synchronized with the cell cycle and stable maintenance in the cell line (as in lysogenization), which proceeds with no viral genome degradation, thus allowing the subsequent restart of virus development. This phenomenon is usually caused by unfavorable growth conditions for the host cell (such as starvation) and is terminated with initiation of either true lysogenization or lytic growth when growth conditions improve. Pseudolysogeny has been known for tens of years; however, its role has often been underestimated. Currently, it is being considered more often as an important aspect of phage-host interactions. The reason for this is mostly an increased interest in phage-host interactions in the natural environment. Pseudolysogeny seems to play an important role in phage survival, as bacteria in a natural environment are starved or their growth is very slow. This phenomenon can be an important aspect of phage-dependent bacterial mortality and may influence the virulence of some bacterial strains.

CRISPR: new horizons in phage resistance and strain identification.

Bacteria have been widely used as starter cultures in the food industry, notably for the fermentation of milk into dairy products such as cheese and yogurt. Lactic acid bacteria used in food manufacturing, such as lactobacilli, lactococci, streptococci, Leuconostoc, pediococci, and bifidobacteria, are selectively formulated based on functional characteristics that provide idiosyncratic flavor and texture attributes, as well as their ability to withstand processing and manufacturing conditions. Unfortunately, given frequent viral exposure in industrial environments, starter culture selection and development rely on defense systems that provide resistance against bacteriophage predation, including restriction-modification, abortive infection, and recently discovered CRISPRs (clustered regularly interspaced short palindromic repeats). CRISPRs, together with CRISPR-associated genes (cas), form the CRISPR/Cas immune system, which provides adaptive immunity against phages and invasive genetic elements. The immunization process is based on the incorporation of short DNA sequences from virulent phages into the CRISPR locus. Subsequently, CRISPR transcripts are processed into small interfering RNAs that guide a multifunctional protein complex to recognize and cleave matching foreign DNA. Hypervariable CRISPR loci provide insights into the phage and host population dynamics, and new avenues for enhanced phage resistance and genetic typing and tagging of industrial strains.

Bacteriophage therapy: potential uses in the control of antibiotic-resistant pathogens.

The use of bacteriophages (phages) to treat bacterial infections, known as phage therapy, has a history substantially longer than that of antibiotics, yet these drugs have been the treatment of choice in the West for over 60 years owing to efficacy, low toxicity and ease of production. Bacteria are becoming increasingly resistant to antibiotics while efforts to discover new agents have drastically reduced. Phages have co-evolved with their hosts over billions of years and have acquired mechanisms to counter bacterial defences such as extracellular biofilm production, which severely reduces the effectiveness of conventional antibiotics. Recent animal and human trials show phages to be safe, well-tolerated agents with a bright future as an alternative to chemical agents.

RinA controls phage-mediated packaging and transfer of virulence genes in Gram-positive bacteria.

Phage-mediated transfer of microbial genetic elements plays a crucial role in bacterial life style and evolution. In this study, we identify the RinA family of phage-encoded proteins as activators required for transcription of the late operon in a large group of temperate staphylococcal phages. RinA binds to a tightly regulated promoter region, situated upstream of the terS gene, that controls expression of the morphogenetic and lysis modules of the phage, activating their transcription. As expected, rinA deletion eliminated formation of functional phage particles and significantly decreased the transfer of phage and pathogenicity island encoded virulence factors. A genetic analysis of the late promoter region showed that a fragment of 272 bp contains both the promoter and the region necessary for activation by RinA. In addition, we demonstrated that RinA is the only phage-encoded protein required for the activation of this promoter region. This region was shown to be divergent among different phages. Consequently, phages with divergent promoter regions carried allelic variants of the RinA protein, which specifically recognize its own promoter sequence. Finally, most Gram-postive bacteria carry bacteriophages encoding RinA homologue proteins. Characterization of several of these proteins demonstrated that control by RinA of the phage-mediated packaging and transfer of virulence factor is a conserved mechanism regulating horizontal gene transfer.

The SPO1-related bacteriophages.

A large and diverse group of bacteriophages has been termed 'SPO1-like viruses'. To date, molecular data and genome sequences are available for Bacillus phage SPO1 and eight related phages infecting members of other bacterial genera. Many additional bacteriophages have been described as SPO1-related, but very few data are available for most of them. We present an overview of putative 'SPO1-like viruses' and shall discuss the available data in view of the recently proposed expansion of this group of bacteriophages to the tentative subfamily Spounavirinae. Characteristics of SPO1-related phages include (a) the host organisms are Firmicutes; (b) members are strictly virulent myoviruses; (c) all phages feature common morphological properties; (d) the phage genome consists of a terminally redundant, non-permuted dsDNA molecule of 127-157 kb in size; and (e) phages share considerable amino acid homology. The number of phages isolated consistent with these parameters is large, suggesting a ubiquitous nature of this group of viruses.